Vibrio cholerae activates proinflammatory response in cultured intestinal epithelial cells. In this study, we demonstrate that V. cholerae O395 infection of intestinal epithelial cells results in the activation of Akt. Inhibition of Akt significantly decreases IL-1alpha, IL-6, and TNF-alpha production in V. cholerae infected Int407 cells. Analysis of the mechanisms of Akt influences on cytokine response demonstrates that Akt promotes NF-kappaB activation. We have extended these findings to show that Akt activation may be regulated by bacterial genes associated with virulence, adherence, or motility. Insertion mutants in the virulence genes coding for CtxA, ToxT, and OmpU of V. cholerae modulate the activation of PI3K/Akt signaling pathway, whereas an aflagellate non-motile mutant (O395FLAN) and a adherent and less motile mutant (O395Y3N/O395Y4N) of V. cholerae both show very significant down-regulation of Akt activity in Int407 cells. Together, these observations indicate that Akt promotes proinflammatory cytokine production by V. cholerae infected human intestinal epithelial cells through its influences on NF-kappaB.