ABSTRACT The carbon isotope composition of Dissolved Inorganic Carbon (δ13CDIC) is a useful tool to study the carbon budget in estuarine settings. Here we present the first set of δ13CDIC data from the Cananéia-Iguape Estuarine Complex (São Paulo, Brazil), a changing environment undergoing freshening at a rate of the order of 770 m3 s-1. We measured δ13CDIC values of water at 15 stations distributed across the area, both during neap and spring tide, in May 2015 and April 2017. The spatio-temporal variability of δ13CDIC suggests a conservative mixing of two water endmembers, freshwater with δ13CDIC = −10 ‰ (V-PDB) and seawater with δ13CDIC = +2.0 ‰ (V-PDB). Linear regression of δ13CDIC vs. salinity predicts the δ13CDIC values of open ocean surface water from the nearby Santos Basin and suggests that the freshwater and seawater endmembers mix at 1:1 proportion in the estuarine complex. Mixing was the dominant process governing the behavior of δ13CDIC values, whereas photosynthesis, degradation of organic matter, and CO2 uptake or outgassing did not play a significant role. In particular, phytoplankton production had no impact on δ13CDIC, suggesting that the ecosystem was exempt from eutrophication. Long-term freshening in the NE sector of the estuary did not significantly impact the δ13C values of DIC. These results underline the need for long-term measurements of δ13CDIC in the study area to contribute to the understanding of the evolution of the estuarine complex, whose freshening trend could be enhanced by extreme precipitation events linked to climate change.