Activation of CD40 by CD40L results in diverse effects on different malignant cells, causing either promotion of survival, growth and resistance to chemotherapy, or induction of cytostasis and apoptosis. The molecular mechanisms underlying CD40-mediated growth regulation and apoptosis induction in cancer cell are not fully understood. In this study, we investigated the role of NF-kappaB activation in CD40-mediated cytotoxicity in cancer cells. The results show that activation of CD40 by recombinant soluble CD40 ligand (rsCD40L) readily induced NF-kappaB activation and blocking NF-kappaB significantly enhanced rsCD40L-induced apoptosis in cancer cells. Importantly, autocrine of TNF-alpha induced by rsCD40L was indispensable for both NF-kappaB activation and cytotoxicity induction, establishing a dual role of autocrine TNF-alpha that constitutes both pro-apoptotic and anti-apoptotic arms of CD40 signaling. Our results indicate that autocrine TNF-alpha-mediated NF-kappaB activation is a determinant for cancer cells' evasion of CD40L-induced cytotoxicity and blocking NF-kappaB may have potential for improve the value of CD40 as an anticancer agent.