We conduct the first stable isotopic and sclerochronological calibration of the bivalve Anomalocardia flexuosa (Linnaeus, 1767) in relation to environmental variables in a subtropical coastal area of southern Brazil. We investigate incremental shell growth patterns and δ18O and δ13C values of modern specimens collected alive from the Laguna Lagoonal System (LLS). As shells of Anomalocardia flexuosa are also the main components of pre-Columbian archaeological shell mounds and middens distributed along the Brazilian coastline, late Holocene archaeological specimens from a local shell mound (Cabeçuda) were selected to compare their stable carbon and oxygen isotopes with those of modern specimens. Shell growth increments, δ18O and δ13C values respond to a complex of environmental conditions, involving, for example, the effects of temperature and salinity. The isotopic information extracted from archaeological specimens from Cabeçuda shell midden in the LLS indirectly indicates that environmental conditions during the late Holocene were different from present day. In particular, intra-shell δ18O and δ13C values of archaeological shells reveal a stronger marine influence at 3 ka cal BP, which is in contrast to the seasonal freshwater/seawater balance that currently prevails at the LLS.