Human (Homo sapiens)micro-RNAs (hsa-miRNAs) regulate virus and host-gene translation, but the biological impact in patientswith human cytomegalovirus (hCMV) infection is notwell defined in a clinically relevantmodel. First, we compared hsa-miRNA expression profiles in peripheral blood mononuclear cells from 35 transplant recipients with and without CMV viremia by using a microarray chip covering 847hsa-miRNAs. This approach demonstrated a set of 142 differentially expressed hsamiRNAs. Next, we examined the effect of each of these miRNAs on viral growth by using human fibroblasts (human foreskin fibroblast-1) infected with the hCMV Towne strain, identifyinga subset of proviral andantiviral hsa-miRNAs. miRNA-target prediction software indicated potential binding sites within the hCMV genome (e.g., hCMV-UL52 and -UL100 [UL¼unique long]) and host-genes (e.g., interleukin-1 receptor, IRF1). Luciferaseexpressing plasmid constructs and immunoblotting confirmed several predicted miRNA targets. Finally, we determined the expression of selected proviral and antiviral hsa-miRNAs in 242 transplant recipients with hCMV-viremia. We measured hsa-miRNAs before and after antiviral therapy and correlated hsa-miRNA expression levels to hCMV-replication dynamics. One of six antiviral hsa-miRNAs showed a significant increase during treatment, concurrent with viral decline. In contrast, six of eight proviral hsa-miRNAs showed a decrease during viral decline. Our results indicate that a complex and multitargeted hsa-miRNA response occurs during CMV replication in immunosuppressed patients. This study provides mechanistic insight and potential novel biomarkers for CMV replication.