The Separation of Light from Darkness is, from the perspective of the Genesis chronology, the first of nine central panels that run along the center of Michelangelo's Sistine Chapel ceiling and which depict scenes from the Book of Genesis. Michelangelo probably completed this panel in the summer of 1512, the last year of the Sistine ceiling project. It is one of five smaller scenes that alternate with four larger scenes that run along the center of the Sistine ceiling. The Separation of Light from Darkness is based on verses 3–5 from the first chapter of the Book of Genesis:

Although in terms of the Genesis chronology it is the first of nine central panels along the Sistine ceiling, the Separation of Light from Darkness was the last of the nine panels painted by Michelangelo. Michelangelo painted the Sistine ceiling in two stages. Between May 1508 and the summer of 1511, he completed the "entrance half" of the Sistine chapel and ended this stage by painting the Creation of Eve and the scenes flanking this central panel. After an idle period of about 6 months, he painted the "altar half," starting with the Creation of Adam, between the winter of 1511 and October 1512.

In the Separation of Light from Darkness, the image of God is framed by four ignudi and by two shields or medallions. The ignudi are young, nude males that Michelangelo painted as supporting figures at each corner of the five smaller narrative scenes along the center of the ceiling. There are a total of 20 ignudi. These figures are all different and appear less constrained within their space than the Ancestors

\ ignudi associated with the Separation of Light from Darkness represent day and night, which echo the theme of light and darkness. For instance, he points out that the ignudo next to God's right hand (on the side of darkness) is stretching as if awakening from sleep in the morning, that the diagonally opposed ignudo below God's knee (on the side of light) is carrying a heavy bundle of oak garlands on his shoulders, representing a daytime activity, and that the ignudo next to God's left arm is falling asleep and signifies nighttime. Pfeiffer and other scholars have also suggested that in Michelangelo's Sistine iconography the ignudi represent angels and that the Bronze Nudes below the ignudi are fallen angels.

Two shields or medallions accompany each set of four ignudi in the five smaller Genesis panels along the center of the Sistine ceiling. They are often described as painted to resemble bronze. Each is decorated with a picture from either the Old Testament or the Book of Maccabees from the Apocrypha. The subjects of the shields are often violent. In the Separation of Light from Darkness the shield above God shows Abraham sacrificing his son Isaac (Genesis 22:9–12), and the one below God shows the prophet Elijah as he is carried up to heaven in a chariot of fire (2 Kings 2:11).

At the center of the composition, God is shown in contrapposto rising into the sky, with arms outstretched separating the light from the darkness. Michelangelo employed in this fresco the challenging ceiling fresco technique of sotto in su ("from below, upward"), which makes a figure appear as if it is rising above the viewer by using foreshortening. The contrapposto pose was also used by Michelangelo in his David (1501-1504). It is reported that Michelangelo painted this fresco in a single giornata, that is, a single working day of approximately eight hours. During Michelangelo's lifetime, this fresco was considered evidence of the painter's technical prowess at its peak. For instance, Giorgio Vasari (1511–1574), Michelangelo's student and biographer, wrote in 1550:

"Furthermore, to demonstrate the perfection of his art and the greatness of God, Michelangelo depicted God dividing the light from darkness in these scenes, where He is seen in all His majesty as He sustains himself alone with open arms with the demonstration of love and creative energy.

According to Vasari, Michaelangelo has "lifted the veil" of darkness typically found in artwork of the Renaissance, employing the skill of chiaroscuro, by combining the intense lights and extreme darks in this symbolic artwork.