Interactions between costimulatory molecules and their receptors are vital for Ag-presenting dendritic cells (DCs) to initiate T cells activation, expansion and their antitumor immune responses. Augmentation of costimulatory signal due to the interaction of DCs and T cells may amplify, sustain and drive diversity of cytotoxic T lymphocytes (CTLs) and consequently enhance the antitumor response. 4-1BBL/4-1BB is such a pair of costimulatory ligand and receptor, playing an important role in the co-stimulation of CTLs. Previously, we demonstrated that DCs transduced with recombinant adenovirus encoding truncated PSMA (tPSMA) and m4-1BBL could induce prostate cancer regression in mouse models. In the present study, we further explored the adjuvant role of 4-1BBL in modulating CTLs activation induced by tPSMA gene-pulsed DCs. The apoptosis and cytotoxicity against tPSMA expressing RM-1 cells of CTLs were determined. Results showed that tPSMA gene-pulsed DCs effectively induced T lymphocyte activation and cytotoxicity, which was enhanced by upregulated expression of 4-1BBL, displaying better cell viability, lower CTLs apoptosis, higher expression anti-apoptotic protein of Bcl-xL and phosphorylation of P38, enhanced NF-κB activation, as well as more IFN-γ production. These results demonstrated that 4-1BBL may play a significant role in the co-stimulation pathway for Ag-presenting DCs-mediated CTLs activity, which might be a beneficial adjuvant factor for DCs-based cancer immunotherapy.