The Aptian Oceanic Anoxic Event 1a (OAE1a, ca.120 Ma ago) is one of the most prominent of a series of geologically brief intervals in the Cretaceous characterized by the deposition of organic carbon–rich sediments. OAEs reflect major perturbations in the global carbon cycle evidenced by sedimentary carbon isotope records. However, the triggering mechanisms for OAEs remain controversial. Here we present a bulk-rock and molecular (marine and terrestrial bio-markers) C isotope record at unprecedented time resolution, from the Cismon section of northern Italy, that shows that OAE1a conditions were reached over a period of several thousands of years through a stepwise perturbation of the carbon cycle. The documented sequence of events is most compatible with a trigger associated with increased CO2 emissions, possibly leading to a doubling of pCO2, which in turn caused larger C isotope fractionation in marine and terrestrial organisms and a major biotic crisis in the calcareous nannoplankton. Our data also show that a release of isotopically light carbon from partial methane hydrate dissociation probably played a minor role in the OAE1a carbon cycle perturbation.