Vibrio vulnificus is part of the natural estuarine microflora and accumulates in shellfish through filter feeding. It is responsible for the majority of seafood-associated fatalities in the United States mainly through consumption of raw oysters. Previously we have shown that a V. vulnificus mutant unable to express PilD, the type IV prepilin peptidase, does not express pili on the surface of the bacterium and is defective in adherence to human epithelial cells (R. N. Paranjpye, J. C. Lara, J. C. Pepe, C. M. Pepe, and M. S. Strom, Infect. Immun. 66:5659-5668, 1998). A mutant unable to express one of the type IV pilins, PilA, is also defective in adherence to epithelial cells as well as biofilm formation on abiotic surfaces (R. N. Paranjpye and M. S. Strom, Infect. Immun. 73:1411-1422, 2005). In this study we report that the loss of PilD or PilA significantly reduces the ability of V. vulnificus to persist in Crassostrea virginica over a 66-h interval, strongly suggesting that pili expressed by this bacterium play a role in colonization or persistence in oysters.