MicroRNAs (miRNAs) are a class of noncoding small RNAs that regulate multiple cellular processes, as well as the replication and pathogenesis of many DNA viruses and some RNA viruses. Examination of cellular miRNA profiles in West Nile virus (WNV)-infected HEK293 and SK-N-MC cells revealed increased expression of multiple miRNA species. One of these miRNAs, Hs_154, was significantly induced not only in WNV-infected neuronal cells in culture but also in the central nervous system tissues of infected mice and, upon transfection, caused a significant reduction in viral replication. Analysis of mRNA transcripts enriched through immunoprecipitation of the RNA-induced silencing complex identified several transcripts that contain seed sequence matches to Hs_154 in their 3' untranslated regions (UTRs). Two of these targets, the CCCTC-binding factor (CTCF) and the epidermal growth factor receptor (EGFR)-coamplified and overexpressed protein (ECOP/VOPP1) proteins display reduced expression in WNV-infected cells, and the 3' UTRs of these transcripts were sufficient to cause downregulation of expression in infected cells or in cells transfected with Hs_154, findings consistent with miRNA targeting of these transcripts. CTCF and ECOP have been shown to be associated with cell survival, implicating miRNA-directed repression of these targets in WNV-induced cell death. Consistent with this hypothesis, expression of these genes in WNV-infected cells results in a reduction in the number of cells undergoing apoptosis. These observations suggest that induction of Hs_154 expression after WNV infection modulates the apoptotic response to WNV and that cellular miRNA expression can be quickly altered during WNV infection to control aspects of the host response.