Heme oxygenase-1 (HO-1) is both beneficial and detrimental to the host in some viral infections by catalyzing the conversion of heme to biliverdin, iron, and carbon monoxide. Simian Virus 40 (SV40) early promoter plays an important role in transforming many cells as it can drive the transcription of large T antigen, which is a potent oncogene. In order to determine the effect of HO-1 on the SV40 early promoter, tumor cells overexpressing HO-1 and HO-1 dominant-negative mutant (glycine143 mutated to histidine) (HO-1G143H) were used. Western blot and HO activity for HO-1/HO-1G143H expression, cell growth, and luciferase activity driven by SV40 promoter were detected in this study. The luciferase activity was suppressed notably in BGC-823 cells transiently overexpressing HO-1, but significantly increased in BGC-823 cells transiently overexpressing HO-1G143H, compared with the mock, respectively. HO-1 overexpression in BGC-823 cells caused the cells containing Blasticidin-resistant gene driven by SV40 promoter to grow slowly under Blasticidin screening, compared with control groups. The luciferase activities were also suppressed in BGC-823, A549, and HepG2 cells stably overexpressing HO-1, and increased in these cell lines stably overexpressing HO-1G143H, compared with the mock, respectively. The results demonstrated that overexpression of HO-1 suppressed transcription driven by SV40 promoter in tumor cells and that HO-1 catalysates might play a major role in the process. Our preliminary results suggested that HO-1 might possess promising counteraction in cell transformation by suppressing SV40 large T-antigen expression, potentially applicable to therapeutic interventions in some virus diseases.