One of the promising strategies for targeting replication of oncolytic adenovirus in tumor cells is to regulate the expression of essential viral genes such as E1a by using tumor- or tissue-specific promoters that are preferentially active in cancer cells. However, this approach may lead to some degree of viral replication in normal cells other than in cancer cells if the viral gene also expresses in normal cells. In this study, we investigated the effect of E1a expression levels on the virus replication ability in human cells. Three vectors, all with mutated E1B55K, were created, one without any promoter controlling the E1a gene and two vectors with the E1a gene being controlled by either its endogenous promoter or a strong CMV promoter. We observed that the CMV promoter-mediated high levels of E1A expression could increase virus replication, resulting in the titers of the E1B55K-mutated virus being even higher than the wild-type virus in some cancer cells. However, the strong CMV promoter could not always enhance virus replication, such as in cancer cells OE33 and OsACL. The results suggest that whether increased E1A levels would enhance E1B55K-mutated virus replication may be also depended on cellular factors or pathways in cancer cells. We also observed that the virus without any promoter for the E1a gene could still express leaky levels of E1A which can lead to viral replication in normal and cancer cells. Future efforts in the development of transcription-controlled oncolytic adenoviruses should focus on how to completely block E1a expression in normal cells.