Delineation of metal uptake routes in aquatic invertebrates is critical for characterizing bioaccumulation dynamics and assessing risks associated with metal exposure. Here we demonstrate that Cu stable isotopic ratios can be manipulated in both exposure media and algae to determine the efflux rate constant (ke) and to estimate Cu assimilation efficiency (AE) from ingested food in a freshwater bivalve (Corbicula fluminea). The Cu AE in Corbicula fed 65Cu-spiked Cryptomonas ozolini was 38%. Copper uptake routes had no significant influence on efflux; ke of 0.004 per day characterized the slowest component of efflux following short-term exposures to 65Cu in water or in both food and water. Incorporation of the physiological parameters for dietary and dissolved uptake as well as rate constants of loss into a bioaccumulation model allowed for assessing the relative contribution of water and food as Cu sources. At [65Cu2+] of 6.7 μg/L, Corbicula accumulated twice as much Cu from diet as from water. In most freshwater systems, the dietary pathway is likely to act as the major Cu uptake route for Corbicula. Extrapolation of our laboratory results to the San Francisco Bay—Delta (California, USA) indicated that our biodynamic model and the laboratory-derived parameters for dietary 65Cu uptake provided a realistic representation of the processes involved in Cu accumulation by the bivalve Corbicula.