Mark Twain Cave — originally McDowell's Cave — is a show cave located near Hannibal, Missouri. It was named for author Mark Twain whose real name was Samuel Langhorne Clemens. Clemens lived in Hannibal from 1839 to 1853, age 4 to 17. It is the oldest operating show cave in the state, giving tours continuously since 1886. Along with nearby Cameron Cave, it became a registered National Natural Landmark in 1972, with a citation reading "Exceptionally good examples of the maze type of cavern development." The cave — as "McDougal's Cave" — plays an important role in Twain's 1876 novel The Adventures of Tom Sawyer and was renamed in honor of the author in 1880.

Geologically Mark Twain Cave and its nearby neighbor Cameron Cave differ from most of the 7,500+ caves found in Missouri. Both are believed to be remnants of a much larger cave system cut apart by a glacier and millions of years of erosion, leading to speculation by geologists and common citizens alike that there may be further undiscovered caves in the Hannibal region. This speculation was heightened in 2006 when the entrance to a previously unknown cave was found during construction of a new elementary school. A major difference is that Mark Twain Cave has a near total lack of speleothems, mineral deposits like stalagmites and stalactites in large open areas. Mark Twain Cave and Cameron Cave instead have a multitude of narrow, winding passages. The caves are made mostly of a soft limestone called Louisiana Lithographic Limestone that is found only in a 35-mile (56 km) area around Hannibal and Louisiana, Missouri. The limestone has been estimated by geologists to be about 350 million years old, while the cave passages were formed some 100 million years ago. Mark Twain Cave covers some 3 miles (4.8 km) with four entrances and 260 passages and has a year-round temperature of 52 °F (11 °C).

While it is possible that prehistoric Native Americans were aware of the cave, the earliest documentation claims that Mark Twain Cave was discovered in the winter of 1819–1820 by a local hunter, Jack Simms, when his dog chased an animal into a small hillside opening south of current-day Hannibal. Upon investigation with torches, he and his brothers found that the small opening led to a large underground labyrinth. The cave proved a popular diversion for mid-19th century Hannibal residents, especially children, including the young Sam Clemens. These childhood explorations would later reappear in five of Mark Twain's books. The proximity to the Mississippi River and its cooling breezes made the small valley between the river bluffs containing the caves a popular site for family picnics and church outings in the summertime.

Pioneering Hannibal physician Joseph Nash McDowell purchased the cave in the late 1840s and used it for several years as a laboratory for medical research on human corpses. His belief that traditional burial that 'stifled the soul of the dead' and that a different type of interment would aid communication between living and dead, led to one of the cave's more notorious episodes, and inspiration for Twain, when McDowell placed his recently deceased child Amanda in a preserving coffin inside the cave work space. Twain's book Life on the Mississippi offered a description of the activities:

In my time the person who owned it [the cave] turned it into a mausoleum for his daughter, age fourteen. The body of this poor child was put in a copper cylinder filled with alcohol, and this suspended in one of the dismal avenues of the cave.

However in 1849, when McDowell learned that locals had been daring each other to break into the cave and open the cylinder, disrespecting his child’s remains, he had the body removed for a more traditional, and safer, burial in the family vault behind the newly-built Missouri Medical College where he worked. A fable of the body being forcibly removed by angry Hannibal citizens, having heard of it from the children who had used the body to enhance their telling of ghost stories, was added to the rumors that swirled around McDowell. Many in St. Louis believed that McDowell also used bodies stolen from local graves for his medical examinations, a not uncommon practice prior to the 20th century. Twain would weave that suspicion into the plot of Tom Sawyer in a grave robbing scene involving Injun Joe.

According to folklore, McDowell used the cave as a secret Confederate weapons storage cache during the American Civil War. McDowell was an ardent Southern supporter, and indeed had a stash of cannons and muskets, previously intended to aid the rebels in his home state of Kentucky in 1846, stockpiled at his St. Louis medical college, as evidenced when an angry mob gathered outside the building, mistakenly accusing McDowell of murder. One of the former Confederates who likely had knowledge of the cave from his war service was the legendary outlaw Jesse James. James had ridden with Quantrill's Raiders and Bloody Bill Anderson throughout the Little Dixie area southwest of Hannibal. In September 1879, two weeks prior to the robbery of a train in Glendale, Missouri, the cave proved a ready and secure hideout for a few days rest. James even signed and dated one of the caves walls using a pencil.

The cave was but a labyrinth of crooked aisles that ran into each other and out again and led nowhere. It was said that one might wander days and nights together through its intricate tangle of rifts and chasms and never find the end of the cave.