We estimated the composition of two food sources for the cultured pearl oyster Pinctada fucata martensii using stable isotopes and stomach content analysis in the coastal areas of the Uwa Sea, Japan. The δ 13C values of oysters (−17.5 to −16.8‰) were intermediate between that of particulate organic matter (POM, −20.2 to −19.1‰) and attached microalgae on pearl cages (−13.0‰). An isotope mixing model suggested that oysters were consuming 78% POM (mainly phytoplankton) and 22% attached microalgae. The attached microalgal composition of the stomach content showed a strong resemblance to the composition of that estimated through the isotope mixing model, suggesting preferential utilization of specific components is unlikely in this species. These results indicate that P. fucata martensii feed on a mixture of phytoplankton and attached microalgae, and that the attached microalgae on pearl cages can serve as an important additional food source.