To accurately assess the behavior and toxicity of silver nanoparticles (AgNPs), it is essential to understand their subcellular distribution and biotransformation. We combined both nanoscale secondary ion mass spectrometry (NanoSIMS) and electron microscopy to investigate the subcellular localization of Ag and in situ chemical distribution in the oyster larvae Crassostrea angulata after exposure to isotopically enriched 109AgNPs. Oyster larvae directly ingested particulate Ag, and in vivo dissolution of AgNPs occurred. The results collectively showed that AgNPs were much less bioavailable than Ag+, and the intracellular Ag was mainly originated from the soluble Ag, especially those dissolved from the ingested AgNPs. AgNPs absorbed on the cell membranes continued to release Ag ions, forming inorganic Ag-S complexes extracellularly, while Ag-organosulfur complexes were predominantly formed intracellularly. The internalized Ag could bind to the sulfur-rich molecules (S-donors) in the cytosol and/or be sequestered in the lysosomes of velum, esophagus, and stomach cells, as well as in the digestive vacuoles of digestive cells, which could act as a detoxification pathway for the oyster larvae. Ag was also occasionally incorporated into the phosphate granules, rough endoplasmic reticulum, and mitochondria. Our work provided definite evidence for the partial sulfidation of AgNPs after interaction with oyster larvae and shed new light on the bioavailability and fate of nanoparticles in marine environment.