Recently we demonstrated that recombinant Cry1Ac protoxin from Bacillus thuringiensis is a potent systemic and mucosal immunogen. In this study we compared the adjuvant effects of Cry1Ac and cholera toxin (CT) for the hepatitis B surface antigen (HBsAg) and bovine serum albumin (BSA). The antibody responses of intestinal secretions and serum were determined by ELISA in Balb/c mice immunized through the intragastric (IG) or intraperitoneal (IP) routes. When HBsAg was administered via IG, the anti-HBsAg intestinal response was not enhanced by either Cry1Ac or CT, whereas via IP Cry1Ac increased the anti-HBsAg intestinal immunoglobulin (Ig)G response and CT increased the intestinal IgA and IgM responses. Serum anti-BSA antibodies increased when BSA was co-administered with CT or Cry1Ac by both routes. Cholera toxin and Cry1Ac co-administered via IP increased the IgG anti-BSA response in fluid of the large intestine and CT also increased the IgA and IgM responses slightly. When co-administered via IP, CT and Cry1Ac did not affect the IgG anti-BSA response of the small intestine significantly. We conclude that Cry1Ac is a mucosal and systemic adjuvant as potent as CT which enhances mostly serum and intestinal IgG antibody responses, especially at the large intestine, and its effects depend on the route and antigen used. These features make Cry1Ac of potential use as carrier and/or adjuvant in mucosal and parenteral vaccines.