The Pottawatomie massacre occurred on the night of May 24–25, 1856, in the Kansas Territory, United States. In reaction to the sacking of Lawrence by pro-slavery forces on May 21, and the telegraphed news of the severe attack on Massachusetts Senator Charles Sumner, John Brown and a band of abolitionist settlers—some of them members of the Pottawatomie Rifles—responded violently. Just north of Pottawatomie Creek, in Franklin County, they abducted and killed five pro-slavery settlers. One teenage son of one of the settlers was also abducted by Brown and his fellow perpetrators, but was ultimately spared.

This soon became the most famous of the many violent episodes of the "Bleeding Kansas" period, during which a state-level civil war in the Kansas Territory was described as a "tragic prelude" to the American Civil War which soon followed. "Bleeding Kansas" involved conflicts between pro- and anti-slavery settlers over whether the Kansas Territory would enter the Union as a slave state or a free state. It has also been described as John Brown's most questionable and controversial act, both to his friends and his enemies. Abolitionist Frederick Douglass described the massacre by John Brown's band as "a terrible remedy for a terrible malady."

John Brown was particularly affected by the sacking of Lawrence, in which the Douglas County Sheriff Samuel Jones on May 21 led a posse that destroyed the presses and type of the Kansas Free State and the Herald of Freedom, Kansas's two abolitionist newspapers, the fortified Free State Hotel, and the house of Charles Robinson. He was the free-state militia commander-in-chief and leader of the "free state" government, established in opposition to the pro-slavery territorial government, based in Lecompton.

A Douglas County grand jury had ordered the attack because the hotel "had been used as a fortress" and an "arsenal" the previous winter, and the "seditious" newspapers were indicted because "they had urged the people to resist the enactments passed" by the territorial governor. The violence against abolitionists was accompanied by celebrations in the pro-slavery press, with writers such as Dr. John H Stringfellow of the Squatter Sovereign proclaiming that pro-slavery forces "are determined to repel this Northern invasion and make Kansas a Slave State; though our rivers should be covered with the blood of their victims and the carcasses of the Abolitionists should be so numerous in the territory as to breed disease and sickness, we will not be deterred from our purpose." Brown was outraged by both the violence of pro-slavery forces and by what he saw as a weak and cowardly response by the anti-slavery partisans and the Free State settlers, whom he described as "cowards, or worse". In addition, two days before the massacre, Brown learned about the caning of abolitionist Charles Sumner by the pro-slavery Preston Brooks on the floor of Congress. Salmon Brown recalled that upon hearing the news, he, his unmarried brothers, and his father went "crazy, crazy," and that "It seemed to be the finishing, decisive touch."

A Free State company under the command of John Brown Jr. set out, and the Osawatomie company joined them. On the morning of May 22, 1856, they heard of the sack of Lawrence and the arrest of Dietzler, George W. Brown, and Jenkins. However, they continued their march toward Lawrence, not knowing whether their assistance might still be needed, and encamped that night near Ottawa Creek. They remained in the vicinity until the afternoon of May 23, at which time they decided to return home.

On May 23, John Sr. selected a party to accompany him on a private expedition. John Jr. objected to their leaving his company, but seeing that his father was unyielding, he consented, telling him, "Father, be careful and commit no rash act." The company consisted of John Brown, four of his sons—Frederick, Owen, Salmon, and Oliver—Thomas Wiener, and James Townsley (who claimed later that Brown had forced him to participate in the incident), whom John had induced to carry the party in his wagon to their proposed field of operations.

Letter of Mahala Doyle to John Brown, when he was in jail awaiting execution, November 20, 1859: "Altho vengence is not mine, I confess, that I do feel gratified to hear that you ware stopt in your fiendish career at Harper's Ferry, with the loss of your two sons, you can now appreciate my distress, in Kansas, when you then and there entered my house at midnight and arrested my husband and two boys and took them out of the yard and in cold blood shot them dead in my hearing, you cant say you done it to free our slaves, we had none and never expected to own one, but has only made me a poor disconsolate widow with helpless children while I feel for your folly. I do hope & trust that you will meet your just reward. O how it pained my Heart to hear the dying groans of my Husband and children if this scrawl give you any consolation you are welcome to it.
NB [postscript] my son John Doyle whose life I begged of (you) is now grown up and is very desirous to be at Charleston [Charles Town] on the day of your execution would certainly be there if his means would permit it, that he might adjust the rope around your neck if Gov. Wise would permit it."

They encamped that night between two deep ravines on the edge of the timber, some distance to the right of the main traveled road. There they remained unobserved until the following evening of May 24. The party left their hiding place sometime after dark and proceeded on their "secret expedition". Late in the evening, they called at the house of James P. Doyle and ordered him and his two adult sons, William and Drury, to go with them as prisoners. Doyle's 16-year-old son, John, who was not a member of the pro-slavery Law and Order Party, was spared after his mother pleaded for his life. The three men were escorted by their captors into the darkness, where Owen Brown and his brother Frederick killed them with broadswords. John Brown Sr. did not participate in the stabbing, instead firing the coup de grâce into the head of the fallen James Doyle.