Genomic data combined with reverse genetic approaches have contributed to the characterization of majorvirulence factors ofVibriospecies; however, these studies have targeted primarily human pathogens. Here, weinvestigate virulence factors in the oyster pathogenVibrio splendidusLGP32 and show that toxicity is correlatedto the presence of a metalloprotease and its correspondingvsmgene. Comparative genomics showed that anavirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloproteasewas confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of thewild type (wt) and avsmdeletion mutant (vsmmutant). The ECPs of the wt induced a strong cytopathic effectwhose severity was cell type dependent, while those of thevsmmutant were much less toxic, and exposure topurified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsmmolecular targets, a proteomic analysis of the ECPs of both LGP32 and thevsmmutant was performed,revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metallo-protease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor ofBacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed byallelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Takentogether, these data demonstrate that Vsm is the major toxicity factor in the ECPs ofV. splendidus.