Mesoproterozoic marine successions worldwide record a shift in average δues from 0 to +3.5 ‰, with the latter value evident in successions younger than 1250 Ma. New carbon isotope data from the ~ 300 to 1270 Ma Dismal Lakes Group, Arctic Canada, provide further insight into this fundamental transition. Data reveal that the shift to higher δ13C values was gradual and marked by occasional excursions to values less than 0 ‰. When compared to records from older and younger marine successions, it is evident that the difference between isotopic minima and maxima increased with time, indicating that the marine system evolved to become isotopically more variable. We interpret these patterns to record an increase in the crustal inventory of organic carbon, reflecting eukaryotic diversification and a change in the locus of organic carbon burial to include anoxic deep marine sites where preservation potential was high. We speculate that the release of O2 to Earth’s surface environments associated with increased organic carbon storage induced irreversible changes in the Mesoproterozoic biosphere, presaging the more extreme environmental and evolutionary developments of the Neoproterozoic.