PI3Kgamma is a phosphoinositide3-kinase predominantly expressed in leucocytes and activated by G-protein-coupled receptors. Cholera toxin (CT) is an AB5 enterotoxin that binds to GM1 ganglioside and catalyzes ADP-ribosylation of Gsalpha. CT is also an effective adjuvant that induces systemic and mucosal immunity. We examined the role played by PI3Kgamma in the mucosal adjuvant activity of CT for epicutaneous vaccines. PI3Kgamma KO and control C57BL/6 mice were immunized three times by transcutaneous application of 50 micrograms of CT with 1 mg ovalbumin (OVA). Control mice developed significant levels of OVA-specific IgA Abs in the serum and in fecal extracts. The adjuvant activity of CT in control mice also resulted in high OVA-specific IgG responses in the serum with IgG1>IgG2b>>IgG2a. Interestingly, the magnitude of OVA-specific serum IgG responses and fecal IgA Abs, were significantly enhanced in PI3Kgamma KO mice. The lack of PI3Kgamma signaling also enhanced Ab responses to CT itself, but did not affect the Th1/Th2 profile of responses. These results suggest that PI3Kgamma plays a regulatory role on immune responses to epicutaneous vaccines with ADP ribosyl-transferase adjuvants.