Recombinant adeno-associated virus (rAAV) is widely used in retinal gene therapy. Enhanced rAAV transduction may be important for better therapeutic effects in some retinal gene therapies. In this study, we examined the effects of adenovirus 5 (Ad5) on retina transduction mediated by rAAV2. Our results provide the first evidence that low levels of either replication-incompetent or conditional replication-competent Ad5 significantly enhance and accelerate transgene expression in human and rat retinal cells. This effect occurs principally at the transcriptional level, rather than through enhanced viral entry or DNA replication. In in vivo analyses with the SD rat, the Balb/c mouse, and the RCS rat, strong enhancement and acceleration of transgene expression, as well as therapeutic effects, were confirmed. Low levels of Ad5 may enhance the utility of rAAV2-mediated transduction strategies in future clinical investigations.