Autophagy is known to have a cytoprotective role under various cellular stresses; however, it also results in robust cell death as an important safeguard mechanism that protects the organism against invading pathogens and unwanted cancer cells. Autophagy is regulated by cell signalling including microRNA (miRNA), a post-transcriptional regulator of gene expression. Here, we show that genetically engineered telomerase-specific oncolytic adenovirus induced miR-7 expression, which is significantly associated with its cytopathic activity in human cancer cells. Virus-mediated miR-7 upregulation depended on enhanced expression of the E2F1 protein. Ectopic expression of miR-7 suppressed cell viability and induced autophagy by inhibiting epidermal growth factor receptor (EGFR) expression. Our results suggest that oncolytic adenovirus induces autophagic cell death through an E2F1-miR-7-EGFR pathway in human cancer cells, providing a novel insight into the molecular mechanism of an anticancer virotherapy.