Cancer therapies that target a single protein or pathway may be limited by their specificity, thus missing key players that control cellular proliferation and contributing to the failure of the treatment. We propose that approaches to cancer therapy that hit multiple targets would limit the chances of escape. To this end, we have developed a bicistronic adenoviral vector encoding both the CDKN2A and p53 tumor suppressor genes. The bicistronic vector, AdCDKN2A-I-p53, supports the translation of both gene products from a single transcript, assuring that all transduced cells will express both proteins. We show that combined, but not single, gene transfer results in markedly reduced proliferation and increased cell death correlated with reduced levels of phosphorylated pRB, induction of CDKN1A and caspase 3 activity, yet avoiding the induction of senescence. Using isogenic cell lines, we show that these effects were not impeded by the presence of mutant p53. In a mouse model of in situ gene therapy, a single intratumoral treatment with the bicistronic vector conferred markedly inhibited tumor progression while the treatment with either CDKN2A or p53 alone only partially controlled tumor growth. Histologic analysis revealed widespread transduction, yet reduced proliferation and increased cell death was associated only with the simultaneous transfer of CDKN2A and p53. We propose that restoration of two of the most frequently altered genes in human cancer, mediated by AdCDKN2A-I-p53, is beneficial since multiple targets are reached, thus increasing the efficacy of the treatment.