Heat shock proteins (HSPs) possess potent antitumor ability to stimulate immune response. We postulated that intratumor injection of oncolytic adenovirus over-expressing HSPs might be able to exert antitumor activity by inducing antitumor immune response in immunocompetent hosts in addition to the oncolytic activity. In this study, two recombinant oncolytic adenoviruses, Ad.CMV.HSP.IRES.E1a and Ad.CMV.IRES.E1a, were constructed with or without the HSP expression cassette. The HSP expression and cytopathic killing effect in mouse B16 melanoma and human PLC/PRF/5 hepatoma cells were measured after in vitro infection of adenoviruses. Survival rate of immunocompetent C57/BL mice was observed following intratumor injection of recombinant adenoviruses in B16 melanoma xenograft models. To detect the evidence of immune responses, hematoxylin and eosin staining and RT-PCR for IFN-gamma expression in tumor tissues were performed. The Ad.CMV.HSP.IRES.E1a induced significantly higher HSP expression level than Ad.CMV.IRES.E1a in both B16 and PLC/ PRF/5 cells. The cytocidal efficacy of Ad.CMV.HSP.IRES.E1a and Ad.CMV.IRES.E1a in PLC/PRF/5 cells was much higher than that in B16 cells, where the two adenoviruses showed similarly very weak oncolytic effect in vitro. However, Ad.CMV.HSP.IRES. E1a improved animal survival rate and exhibited more potent anti-tumor efficiency than Ad.CMV.IRES.E1a in B16 xenograft models. The enhanced efficacy might be mainly attributed to the HSP-mediated immune activity, as evidenced by the up-regulated expression of IFN-gamma and local heavier intratumor inflammatory cell infiltration. These results indicated that the recombinant oncolytic adenovirus over-expressing HSPs possessed powerful in vivo anti-tumor efficacy and might be a valuable approach for cancer immune gene therapy.