Porcine reproductive and respiratory syndrome virus (PRRSV) has recently caused heavy economic losses in swine industry worldwide. Current vaccination strategies only provide a limited protective efficacy, thus immune modulators are being considered to enhance the effectiveness of PRRSV vaccines. In this study, the recombinant adenoviruses expressing porcine CD40 ligand (CD40L) and GP3/GP5 of PRRSV were constructed and the immune responses were examined in pigs. The results showed that rAd-CD40L-GP35 (co-expressing CD40L and GP3-GP5) or rAd-GP35 (expressing GP3-GP5) plus rAd-CD40L (expressing CD40L) could provide significant higher specific anti-PRRSV ELISA antibody and neutralizing antibody. And the levels of proliferative responses of peripheral blood mononuclear cells (PBMC), IFN-γ and IL-4 were markedly increased in rAd-CD40L-GP35 and rAd-CD40L plus rAd-GP35 groups than those in rAd-GP35 group. Following homologous challenge with Chinese isolate of the North-American genotype of PRRSV, pigs inoculated with recombinant rAd-CD40L-GP35 and rAd-CD40L plus rAd-GP35 showed lighter clinical signs and lower viremia, as compared to those in rAd-GP35 group. It indicated that porcine CD40L could effectively increase humoral and cell-mediated immune responses of GP3 and GP5 of PRRSV. Porcine CD40L might be used as an attractive adjuvant or immunotargeting strategies to enhance the PRRSV subunit vaccine responses in swine.