The mechanisms of protection elicited by smallpox vaccine in humans have not been delineated. Dendritic cells (DCs) are the most potent of professional antigen presenting cells which on activation modulate both innate and adaptive immune responses. Immunogenic proteins of vaccinia virus have been identified with protein microarray technology. We have investigated the effect of three most immunogenic proteins, D8L, D13L, and H3L on human monocyte-derived DCs with a view to understand the mechanisms underlying the immunogenicity of these proteins. Our data show that all three proteins activate and mature DCs, induce secretion of cytokines IL-12p70, IL-10, TNF-alpha, IL-6 from DCs, and rVV protein-loaded DCs induce secretion of IFN-gamma and proliferation of T cells with selective expansion of effector memory T cells. Overall D13L and H3L proteins are more immunogenic than D8L. Taken together data suggest that these peptides are highly immunogenic and can modulate an effective innate and adaptive response in humans in vitro.