The objective of this study was to assess the clinical efficacy of genetically engineered recombinant human adenovirus type 5 (rhAd5) plus transcatheter arterial chemoembolization in patients with unresectable hepatocellular carcinoma (HCC). Data from two groups of patients with unresectable HCC were retrospectively reviewed. One group included 149 patients treated with rhAd5 injection, and the other included 150 control patients without gene therapy. Differences in short-term treatment effectiveness and adverse events were recorded and compared between the two groups. Our results indicated that for patients with higher tumor staging in the treatment group, the overall response rate and the disease control rate were higher than those in the control group, but not statistically significant (P > 0.05). The total progression free survival (PFS) and overall survival (OS) were significantly longer in the treatment group than the control group (240 vs. 196 days, P < 0.05; and 1,526 vs. 1,236 days, P = 0.000; respectively). The overall incidence rate of treatment-related adverse effects was similar (P > 0.05). No serious complications were observed. In conclusion, this study suggests that rhAd5 is a safe, effective gene therapy that prolongs the PFS and OS time of patients with unresectable HCC.