Cholera toxin (CT), the enterotoxin of Vibrio cholerae, is a potent mucosal and systemic immunogen and adjuvant. The precise mechanism of the adjuvanticity of CT is poorly understood. Our previous work has showed that CT up-regulates B7.2, but not B7.1 expression on macrophages, and thus increases their co-stimulatory activity. In the current study, the effects of CT on macrophage co-stimulatory cytokine production were investigated. Bone marrow macrophages were generated by culturing bone marrow cells with macrophage colony-stimulating factor. CT treatment increased endotoxin-stimulated macrophage IL-10, IL-6, and IL-1beta production, whereas it decreased IL-12, TNF-alpha and nitric oxide production. Antibody blocking experiments showed that CT inhibition of IL-12 and TNF-alpha production was mediated by increased IL-10 production, in that addition of anti-IL-10 monoclonal antibody abrogated CT inhibition. The decrease in nitric oxide production was in turn secondary to inhibition of TNF-alpha production. Taken together, our study demonstrated that CT has differential effects on various macrophage co-stimulatory cytokines, effects that are likely to contribute to its adjuvanticity.