The fish assemblage structure was analyzed along an estuarine gradient of a small macrotidal estuary (the Canche, France). Fishes were collected every two months between May 2006 and July 2007 from 12 sampling stations using a 1.5-m beam trawl with a 5 mm mesh size in the cod end. To complement this information, sampling was also performed using 15-m fyke nets (8 mm mesh size in the cod end). For each sample, abiotic (temperature, salinity, pH, oxygen, turbidity, river flow, wind speed and depth) and biotic (macro crustacean species abundances) were recorded. Throughout the study, 28 fish species belonging to 20 families were collected. Fish catches were dominated by juveniles, especially Young-Of-the-Year (YOY) for the majority of the species. According to the Index of Relative Importance (IRI), common goby Pomatoschistus microps, flounder Platichtys flesus, sprat Sprattus sprattus, sea-bass Dicentrarchus labrax and plaice Pleuronectes platessa were the most abundant species, together accounting for 99.2% of the total IRI. Estuarine residents (ER = 66.2%) and marine juvenile migrants species (MJ = 31.4%) were the most important ecological guilds. The structure of the fish assemblage and its relationship to environmental variables was examined using multivariate techniques. Cluster and non-metric multidimensional scaling (nMDS) analysis defined six distinct groups in the Canche estuary, which are discriminated by specific species (SIMPER). Spatio-temporal variations in fish assemblage structure reflect the density peaks of the most abundant species. Spearman rank correlations and canonical correspondence analysis (CCA) showed that among the ten environmental variables examined, temperature, salinity and Crangon crangon (a potential predator for YOY fish or prey for older ones) are the three most important factors influencing fish species richness and abundances. Our observations reinforce the idea that certain fish species may have different life history styles in different geographic areas. The present study highlights the necessity of a better knowledge of the connectivity between estuaries and adjacent marine areas. The Canche constitutes an important ecosystem for fishes and as it is subject to little anthropogenic disturbance; its ichthyofauna can be viewed as a reference or normal assemblage for small temperate macrotidal estuaries.