Leishmania, a protozoan parasite, lives and multiplies as amastigote within macrophages. It is proposed that the macrophage expressed CD40 interacts with CD40 ligand on T cells to induce IFN-gamma, a Th1-type cytokine that restricts the amastigote growth. Here, we demonstrate that CD40 cross-linking early after infection resulted in inducible nitric oxide synthetase type-2 (iNOS2) induction and iNOS2-dependent amastigote elimination. Although CD40 expression remained unaltered on L. major-infected macrophages, delay in the treatment of macrophages or of mice with anti-CD40 antibody resulted in significant reduction in iNOS2 expression and leishmanicidal function suggesting impaired CD40 signaling in Leishmania infection. The inhibition of CD40-induced iNOS2 expression by SB203580, a p38-mitogen activated protein kinase (p38MAPK)-specific inhibitor, and the reversal of the inhibition by anisomycin, a p38MAPK activator, suggested a crucial role of p38MAPK in CD40 signaling. Indeed, the CD40-induced p38MAPK phosphorylation, iNOS2 expression and anti-leishmanial function were impaired in Leishmania-infected macrophages but were restored by anisomycin. Anisomycin's effects were reversed by SB203580 emphasizing the role of p38MAPK in CD40-induced iNOS2-dependent leishmanicidal function. Anisomycin administration in L. major-infected BALB/c mice resulted in significant reduction in the parasite load and established a host-protective Th1-type memory response. Also implicated in these findings is a scientific rationale to define novel anti-parasite drug targets and to bypass the problem of drug resistance.