Vertical distributions of dissolved organic carbon (DOC) were determined in different seasons in the southern Adriatic and Tyrrhenian Seas to study the role of stratification in DOC patterns. These two seas are located at similar latitude but differ in extents of vertical stratification. Stratification affects DOC dynamics in both basins, but with interesting differences. In the Tyrrhenian Sea, where the upper water column was stratified during all cruises, DOC showed high surface layer values without substantial seasonality. By contrast, in the southern Adriatic Sea, the seasonal cycle of stratification forced opposite trends in the 0–50 m and 50–800 m stocks, with DOC removal from the upper 50 m associated with DOC increase below 50 m. Regarding DOC export via deep water formation in the southern Adriatic Sea, we estimate that 0.19 Tg C yr−1 was exported to the 50–800 m layer by convective overturn, while 0.85–1.19 Tg C yr−1 was sequestered below 1000 m due to a continental shelf pump mechanism. We hypothesize that enhanced stratification associated with a warmer ocean could further increase DOC concentrations in the mixed layer, changing the role of DOC in the oceanic carbon cycle.