The ultrafast photoinduced ring-opening of 1,3-cyclohexadiene constitutes a textbook example of electrocyclic reactions in organic chemistry and a model for photobiological reactions in vitamin D synthesis. Although the relaxation from the photoexcited electronic state during the ring-opening has been investigated in numerous studies, the accompanying changes in atomic distance have not been resolved. Here we present a direct and unambiguous observation of the ring-opening reaction path on the femtosecond timescale and subångström length scale using megaelectronvolt ultrafast electron diffraction. We followed the carbon–carbon bond dissociation and the structural opening of the 1,3-cyclohexadiene ring by the direct measurement of time-dependent changes in the distribution of interatomic distances. We observed a substantial acceleration of the ring-opening motion after internal conversion to the ground state due to a steepening of the electronic potential gradient towards the product minima. The ring-opening motion transforms into rotation of the terminal ethylene groups in the photoproduct 1,3,5-hexatriene on the subpicosecond timescale.