MicroRNAs (miRNAs) are small RNAs, 19-23 nucleotides in length, which regulate a variety of cellular processes. Human cytomegalovirus (HCMV) encodes only one intronic miRNA: human cytomegalovirus microRNA UL36 (hcmv-miR-UL36). In this study, we found that over-expression of hcmv-miR-UL36 resulted in a more than threefold increase in HCMV DNA synthesis at 24 h post infection. Fifteen putative targets of hcmv-miR-UL36 were identified using hybrid PCR, one being the HCMV UL138 gene that has previously been identified as a novel latency-associated determinant of HCMV infection. Down-regulation of UL138 expression by hcmv-miR-UL36 was validated using luciferase reporter assays and Western blot analysis in HEK293 cells. In the presence of hcmv-miR-UL36, we observed a 74.6 percent decrease in luciferase activity and a 46.2 percent decrease in HCMV UL138 protein expression. Our results indicate that hcmv-miR-UL36 may be a viral miRNA contributing to HCMV replication.