Oncolytic adenoviruses represent a novel class of anticancer agents. Their efficacy in killing cancer cells is variable, suggesting that there is room for improvement. Host miRNAs have been shown to play important roles in susceptibility of cells to replication of different viruses. This study investigated if adenovirus replication in human prostate cancer cells is influenced by host cell miRNA expression. To this end, human miRNA expression in response to adenovirus infection was analyzed, and functional screens for lytic adenovirus replication were performed using synthetic miRNA mimic and inhibitor libraries. Adenovirus infection generally reduced miRNA expression. On top of this nonspecific interference with miRNA biogenesis, a set of miRNAs, including in particular miR-222, was found specifically reduced. Another set of miRNAs was found to promote adenovirus-induced death of prostate cancer cells. In most cases, this did not stimulate adenovirus propagation. The exception was miR-26b. Overexpression of miR-26b inhibited adenovirus-induced NF-κB activation, augmented adenovirus-mediated cell death, increased adenovirus progeny release, and promoted adenovirus propagation and spread in several human prostate cancer cell lines. This suggests that miR-26b is particularly useful to be combined with oncolytic adenovirus for more effective treatment of prostate cancer.