The present study was designed to investigate the synergistic inhibitory effects on hepatocellular carcinoma with recombinant human adenovirus Aspp2 (Aspp2-ad) and oxaliplatin via p53-independent pathway in vitro and in vivo. After being treated with Aspp2-ad and/or oxaliplatin for 24-48 h, HepG2P53-/- and Hep3B cells showed a significant growth inhibition compared with vehicle control. Combination group showed a synergetic effect, the inhibitory rates were all above 80% at 48 h point in HepG2P53-/- and Hep3B cells. The apoptotic cell numbers of Aspp2-ad and/or oxaliplatin treatment groups were increased remarkably, especially for the combined therapy group in the liver cancer cells. The Hep3B xenograft experiment also showed similar inhibition of Aspp2-ad and/or oxaliplatin to the in vitro experiment. H&E results showed that combination group had the least mitotic indexes and the most necrosis. The immunohistochemistry results showed that PCNA, CD31 expression decreased greatly in treatment groups. These results suggested that Aspp2-ad might inhibit proliferation and vascular growth of hepatocarcinoma. Aspp2 induced apoptosis protein expression in Aspp2-ad and combination groups, the Aspp2, Bax and activation of caspase-3 expression increased greatly both in vitro and in vivo. But interestingly, the autophagy proteins showed different responses not only in HepG2P53-/- and Hep3B cells but also in vitro and in vivo. We found that Aspp2-ad downregulated the p-ERK, p-STAT3 expression, the synergistic effects were observed in combination group, while there was not response of mTOR to Aspp2-ad. In conclusion, Aspp2-ad, in P53-independent manner, regulated ERK and STAT3 signal moleculars to inhibit hepatocarcinoma in coordination with oxaliplatin by influencing the protein expression of proliferation, apoptosis, autophagy and vascular growth. Aspp2-ad has the potential to be developed in gene therapy for HCC, especially for P53 deletion or mutation in HCC.