Secretory tumor necrosis factor-alpha (sTNF-alpha) is known to mediate activation-induced cell death (AICD). However, the role of tmTNF-alpha in AICD is still obscure. Here, we demonstrated that tmTNF-alpha expression significantly increased accompanied with enhanced apoptosis during AICD in Jurkat and primary human T cells. Knockdown or enhancement of tmTNF-alpha expression in activated T cells suppressed or promoted AICD, respectively. Treatment of activated T cells with exogenous tmTNF-alpha significantly augmented AICD, indicating that tmTNF-alpha as an effector molecule mediates AICD. As tmTNF-alpha can function as a receptor, an anti-TNF-alpha polyclonal antibody was used to trigger reverse signaling of tmTNF-alpha. This antibody treatment upregulated the expression of Fas ligand, TNF-related apoptosis-inducing ligand and tmTNF-alpha to amplify AICD, and promoted activated T cells expressing death receptor 4, TNF receptor (TNFR) 1 and TNFR2 to enhance their sensitivity to AICD. Knockdown of TNFR1 or TNFR2 expression totally blocked tmTNF-alpha reverse signaling increased sensitivity to sTNF-alpha- or tmTNF-alpha-mediated AICD, respectively. Our results indicate that tmTNF-alpha functions as a death ligand in mediation of AICD and as a receptor in sensitization of activated T cells to AICD. Targeting tmTNF-alpha in activated T cells may be helpful in facilitating AICD for treatment of autoimmune diseases.