Stable isotopes (delta13C and delta15N) were used to analyse the food web downstream of the largest estuary on the French coast: the Gironde. The different sources of organic matter supporting the most abundant and commercially important fish species were determined, as well as habitat connectivity for fish. Stable isotope analysis was performed in different producers (marine, freshwater and local sources), primary consumers (zooplankton and macrozoobenthos) and nine fish species (Alosa alosa, Engraulis encrasicolus, Sprattus sprattus, Liza ramada, Pomatoschistus minutus, Platichthys flesus, Solea solea, Dicentrarchus punctatus and Argyrosomus regius) in three habitats of the downstream area of the estuary in June–July 2012. All sources and invertebrates had significantly different isotopic signatures in different habitats. Only sole, S. solea, presented distinct dual isotopic signatures, indicating a higher feeding location fidelity, no other fish species showed significant differences in isotopic signatures. This overlap was interpreted as evidence that fish had not been feeding exclusively in the habitat where they were collected, instead ingesting food with different isotopic signatures, reflecting high habitat connectivity for these fish. As the base of the fish food web significantly differed among habitats, the present study indicated the suitability of stable isotopes in tracing fish movements and their fidelity/connectivity for habitats separated by less than 10 km, particularly estuarine habitats without salinity differences but located on opposite banks. The SIAR mixing model estimations of organic matter contribution to fish diets in the Gironde estuary were quite similar for the fish species investigated. The major organic source was marine-derived POM, with contributions >75 % for each species. Freshwater and local POM (generally indicated as the sources structuring estuarine food webs) contributed little to the overall fish food webs in the Gironde estuary. Only flounder, P. flesus, and shad, A. alosa, migratory amphihaline species, utilised freshwater POM in greater proportion than marine. The observed low freshwater POM-high marine POM contribution to the fish food web seems to be explained by the reduced intertidal surface of the system. This characterization of the trophic base and habitat connectivity for the most important Gironde estuary fish provides a novel insight for future management of the estuary, especially in the current context of global change.Read less <