Van't Hoff was born at Rotterdam in the Netherlands, the son of a physician. He studied at Delft Polytechnic and the University of Leiden before going abroad to work with August Kekulé in Bonn (1872) and with Charles Adolphe Wurtz in Paris (1874), where he met Joseph-Achille Le Bel. In 1878 he was appointed to the Amsterdam chair of chemistry where he remained until moving to the University of Berlin in 1896.

In 1874 van't Hoff published a paper entitled A Suggestion Looking to the Extension into Space of the Structural Formulas at Present Used in Chemistry, which effectively created a new branch of science – stereochemistry. The problem began with the discovery of optically active compounds. Louis Pasteur later established the asymmetry of crystals of tartaric acid: some would rotate polarized light to the right and others to the left. This was explained by the actual asymmetry of the crystal: the crystals were mirror images of each other. Pasteur thought that the molecules themselves were asymmetric but could offer no proof. This would explain the further problem of the optical activity of noncrystalline solutions. Van't Hoff solved these problems and offered an account of molecular asymmetry by concentrating on the structure of the carbon atom, newly established by Kekulé. He announced (1874) that the four chemical bonds that carbon can form are directed to the corners of a tetrahedron. With this structure, certain molecules can have left- and right-handed isomers, which have opposite effects on polarized light. It also explained why certain isomers do not occur.

Van't Hoff's account of molecular structure was attacked by Hermann Kolbe but a similar theory was put forward simultaneously by Joseph-Achille Le Bel, independently of van't Hoff. Despite the hostility, his ideas were soon vindicated by Emil Fischer's researches into sugars in the 1880s.

Major contributions were also made by van't Hoff to the thermodynamics and kinetics of solutions. Many of these results are reported in his book Etudes de dynamique chimie (1884; Studies in Chemical Kinetics). He had the central insight in 1886 that there is a similarity between solutions and gases provided that osmotic pressure is substituted for the ordinary pressure of gases, and derived laws for dilute solutions similar to those of Robert Boyle and Joseph Gay-Lussac for gases. This fundamental result could be used to determine the molecular weight of a substance in solution.

In 1901 van't Hoff was awarded the first Nobel Prize for chemistry.