The CD40-CD154 system controls various aspects of the host inflammatory response in models of cellular and humoral immunity. Recently, we described a role for CD40 in the innate immune response in polymicrobial sepsis. However, recent data suggests that CD40 maybe activated by CD154 or directly via bacterial heat shock protein (HSP) 70. Therefore, we decided to test the mechanism of CD40 activation in murine polymicrobial sepsis. Wild-type (WT), CD40, and CD154 underwent cecal ligation and puncture (CLP). Compared with WT mice, CD40 had improved survival in association with attenuated production of IL-12, TNF-alpha, and IL-6. In contrast, CD154 mice behaved similar to WT mice with regard to mortality and cytokine production. The differential response of CD40 and CD154 mice to CLP was not due to a general attenuated response to inflammatory stimuli, as all three strains had similar survival after LPS administration, and CD40 macrophages had normal production of IL-12 in response to lipopolysaccharide. In contrast, CD40 macrophages had attenuated IL-12 production in response to Escherichia coli HSP70 (DnaK). Furthermore, intraperitoneal administration of DnaK resulted in a 4-fold increase in IL-12 in WT mice, which was absent in CD40 mice. This data demonstrates CD154-independent CD40 activation in polymicrobial sepsis and suggests that bacterial HSP70 is capable of stimulating CD40 in vitro and in vivo.