First, he enrolled at Delft University of Technology in September 1869, and studied until 1871, when he passed his final exam on 8 July and obtained a degree of chemical technologist.

They had two daughters, Johanna Francina (1880–1964) and Aleida Jacoba (1882–1971), and two sons, Jacobus Henricus van 't Hoff III (1883–1943) and Govert Jacob (1889–1918).

In 1874, he accounted for the phenomenon of optical activity by assuming that the chemical bonds between carbon atoms and their neighbors were directed towards the corners of a regular tetrahedron.

Three months before his doctoral degree was awarded, Van 't Hoff published this theory, which today is regarded as the foundation of stereochemistry, first in a Dutch pamphlet in the fall of 1874, and then in the following May in a small French book entitled La chimie dans l'espace.

In these early years his theory was largely ignored by the scientific community, and was sharply criticized by one prominent chemist, Hermann Kolbe.

Kolbe wrote: "A Dr. J. H. van 't Hoff of the Veterinary School at Utrecht has no liking, apparently, for exact chemical investigation.

He has considered it more convenient to mount Pegasus (apparently borrowed from the Veterinary School) and to proclaim in his ‘La chimie dans l’espace’ how, in his bold flight to the top of the chemical Parnassus, the atoms appeared to him to be arranged in cosmic space."

However, by about 1880, support for Van 't Hoff's theory by such important chemists as Johannes Wislicenus and Viktor Meyer brought recognition.

In 1887, he and German chemist Wilhelm Ostwald founded an influential scientific magazine named Zeitschrift für physikalische Chemie ("Journal of Physical Chemistry").

He was awarded the Helmholtz Medal of the Prussian Academy of Sciences (1911), and appointed Knight of the French Legion of Honour (1894) and Senator in the Kaiser-Wilhelm-Gesellschaft (1911).