Porcine reproductive and respiratory syndrome virus (PRRSV) has recently caused catastrophic losses in swine industry worldwide. Current vaccination strategies only provide a limited protection against PRRSV infection. This study was aimed to construct the recombinant adenovirus co-expressing GP3 and GP5 of highly pathogenic PRRSV fused with swine granulocyte-macrophage colony stimulating factor (GM-CSF) (rAd-GF35), and to detect the immune response in mice and pigs. The results showed that the rAd-GF35 could induce significantly higher PRRSV-specific neutralizing antibodies than the recombinant adenovirus only expressing GP3 and GP5 (rAd-GP35). Moreover, the fusion of GM-CSF markedly increased the secretion of IFN-gamma and IL-4 in PRRSV-stimulated mice lymphocytes culture and pigs sera. Following challenge with PRRSV, piglets inoculated with recombinant rAd-GF35 had lighter clinical signs, lower viremia and less gross lesion of lungs, as compared to that of rAd-GP35 immunized group. It demonstrated that GM-CSF fused with GP3 and GP5 of PRRSV could significantly enhance the humoral and cellular immune responses and provide protection against PRRSV challenge in pigs. The recombinant adenovirus rAd-GF35 might be an attractive candidate vaccine for the prevention and control of highly pathogenic PRRSV infection.