Oncolytic virotherapy can selectively destroy cancer cells and is a potential approach in cancer treatment. A strategy to increase tumor-specific selectivity is to control the expression of a key regulatory viral gene with a tumor-specific promoter. We have previously found that cyclin E expression is augmented in cancer cells after adenovirus (Ad) infection. Thus, the cyclin E promoter that is further activated by Ad in cancer cells may have unique properties for enhancing oncolytic viral replication. We have shown that high levels of viral E1a gene expression are achieved in cancer cells infected with Ad-cycE, in which the endogenous Ad E1a promoter was replaced with the cyclin E promoter. Ad-cycE shows markedly selective oncolytic efficacy in vitro and destroys various types of cancer cells, including those resistant to ONYX-015/dl1520. Furthermore, Ad-cycE shows a strong capacity to repress A549 xenograft tumor growth in nude mice and significantly prolongs survival. This study suggests the potential of Ad-cycE in cancer therapy and indicates the advantages of using promoters that can be upregulated by virus infection in cancer cells in development of oncolytic viruses. Key messages: Cyclin E promoter activity is high in cancer cells and enhanced by adenovirus infection. Cyclin E promoter is used to control the E1a gene of a tumor-specific oncolytic adenovirus. Ad-cycE efficiently targets cancer cells and induces oncolysis. Ad-cycE significantly repressed xenograft tumor and prolonged survival.