Dissolved inorganic carbon (DIC), its stable isotope composition (δ 13 C-DIC) and ancillary parameters of the water column were investigated in a eutrophic tropical marine-dominated estuary surrounded by a large urban area (Guanabara Bay, Rio de Janeiro, Brazil). Most negative δ 13 C-DIC signatures (down to −6.1‰) were found in polluted regions affected by direct sewage discharges where net heterotrophy induces high partial pressure of CO 2 (pCO 2) and DIC concentrations. Keeling plot was applied to this polluted region and determined the δ 13 C-DIC sewage signature source of −12.2‰, which is very consistent with isotopic signature found in wastewater treatment plans. These negative δ 13 C-DIC signatures (i.e., DIC depleted in 13 C) were restricted to the vicinity of urban outlets, whereas in the largest area of the bay δ 13 C-DIC signatures were more positive (i.e., DIC enriched in 13 C). The most positive δ 13 C-DIC signatures (up to 4.6‰) were found in surface waters dominated by large phytoplankton blooms, with positive correlation with chlorophyll a (Chl a). In the largest area of the bay, the preferential uptake of the lighter stable carbon isotope (12 C) during photosynthesis followed the Rayleigh distillation, and appeared as the most important driver of δ 13 C-DIC variations. This reveals an important isotopic fractionation (ε) by phytoplankton due to successive algal blooms that has turned the remaining DIC pool enriched with the heavier stable carbon isotope (13 C). The calculated diel apparent ε showed higher values in the morning (18.7‰-21.6‰) and decreasing in the afternoon (6.8‰-11.1‰). ε was positively correlated to the pCO 2 (R 2 = 0.88, p = 0.005) and DIC concentrations (R 2 = 0.73, p = 0.02), suggesting a decline in carbon assimilation efficiency and decreasing uptake of the lighter carbon under CO 2 limiting conditions. The eutrophic coastal waters of Guanabara Bay have δ 13 C-DIC signatures well above that found in estuaries, shelf and ocean waters worldwide.