4′,5,7-trihydroxy-3′,5′-dimethoxyflavone (tricin), derived from Sasa albo-marginata, has been reported to suppress significantly human cytomegalovirus (HCMV) replication in human embryonic lung (HEL) fibroblast cells. However, the target protein of tricin remains unclear. This study focused on the anti-HCMV activity of tricin in terms of its binding affinity to cyclin-dependent kinase 9 (CDK9). A molecular docking study predicted that tricin binds well to the ATP-binding site of CDK9. Experimental measurements then revealed that tricin inhibits the kinase activity of CDK9 and affects the phosphorylation of the carboxy-terminal domain of RNA polymerase II. Based on these results, we conclude that CDK9 is one of the target proteins of tricin. We also found that tricin possesses anti-HCMV activity with no cytotoxicity against HEL cells.