Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important contagious agents of swine in the world. PRRSV infection poses a challenge to current vaccination strategies. In this study, three replication-defective adenovirus recombinants were developed as potential vaccine against PRRSV in a mouse model. Three groups of BALB/c mice (24 mice per group) were inoculated subcutaneously twice at 2-week intervals with the recombinants expressing PRRSV GP5 (rAd-GP5), M (rAd-M), and M-GP5 fusion protein (rAd-M-GP5). Two additional groups were injected with wild-type adenovirus (wtAd) or PBS as control. The results showed that the mice inoculated with recombinant adenoviruses developed PRRSV-specific antibodies, cellular immune response by 2 weeks post second inoculation. However, only mice immunized with recombinant adenovirus rAd-M-GP5 developed significantly higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to mice immunized with rAd-M or rAd-GP5 alone. It was also found that mice immunized with rAd-M-GP5 were primed for significant higher levels of anti-PRRSV CTL responses than mice immunized with rAd-M. Mice receiving rAd-GP5 also mounted PRRSV-specific response, but levels were lower. It suggested that the recombinant adenovirus expressing M-GP5 fusion protein might be an attractive candidate vaccine to be tested for preventing PRRSV infection.