The induction of macrophage death is regarded as a potential mechanism by which components secreted by Clostridium septicum are used to evade the innate immune response and cause tissue damage. This study aimed to determine the effect of partially purified fractions of extracellular proteins secreted by C. septicum on the death of mouse peritoneal macrophages. Elicited mouse peritoneal macrophages were incubated with partially purified fractions of proteins secreted by C. septicum into culture medium. After incubation, we found that the protein fraction with a molecular weight ≥ 100 kDa caused significant cell death in macrophages, changed cell morphology, increased markers of apoptosis and autophagy, and increased the expression (protein and mRNA) of IL-1 and TNFα. Our data suggest that the proteins secreted by C. septicum (MW ≥100kDa) induce cell death in macrophages by promoting autophagy-triggered apoptosis. This may contribute to our understanding of the molecular mechanism of immune evasion by C. septicum at the infection site.