In this study we show Yersinia pseudotuberculosis secretes membrane vesicles (MVs) that contain different proteins and virulence factors depending on the strain. Although MVs from Y. pseudotuberculosis YPIII and ATCC 29833 had many proteins in common (68.8% of all the proteins identified), those located in the outer membrane fraction differed significantly. For instance, the MVs from Y. pseudotuberculosis YPIII harbored numerous Yersinia outer proteins (Yops) while they were absent in the ATCC 29833 MVs. Another virulence factor found solely in the YPIII MVs was the cytotoxic necrotizing factor (CNFy), a toxin that leads to multinucleation of host cells. The ability of YPIII MVs to transport this toxin and its activity to host cells was verified using HeLa cells, which responded in a dose-dependent manner; nearly 70% of the culture was multinucleated after addition of 5 µg/ml of the purified YPIII MVs. In contrast, less than 10% were multinucleated when the ATCC 29833 MVs were added. Semi-quantification of CNFy within the YPIII MVs found this toxin is present at concentrations of 5 ~ 10 ng per µg of total MV protein, a concentration that accounts for the cellular responses seen.