De Sitter, the son of a judge from Leiden in the Netherlands, studied mathematics and physics at the University of Groningen, his interest in astronomy being aroused by Jacobus Kapteyn. After serving at the Cape Town Observatory from 1897 to 1899 and, back at Groningen, as assistant to Kapteyn from 1899 to 1908, he was appointed to the chair of astronomy at the University of Leiden. He also served as director of Leiden Observatory from 1919 to 1934.

De Sitter is remembered for his proposal in 1917 of what came to be called the de Sitter universe in contrast to the Einstein universe. Einstein had solved the cosmological equations of his general relativity theory by the introduction of the cosmological constant, which yielded a static universe. But de Sitter, in 1917, showed that there was another solution to the equations that produced a static universe if no matter was present. The contrast was summarized in the statement that Einstein's universe contained matter but no motion while de Sitter's involved motion without matter.

The Russian mathematician Alexander Friedmann in 1922 and the Belgian George Lemaître independently in 1927 introduced the idea of an expanding universe that contained moving matter. It was then shown in 1928 that the de Sitter universe could be transformed mathematically into an expanding universe. This model, the Einstein–de Sitter universe, comprised normal Euclidean space and was a simpler version of the Friedmann–Lemaître models in which space was curved.

De Sitter also worked on celestial mechanics and stellar photometry. He spent much time trying to calculate the mass of Jupiter's satellites from the small perturbations in their orbits. The results were published in 1925 in his New Mathematical Theory of Jupiter's Satellites.