Human cytomegalovirus (HCMV) infection has been shown to contribute to vascular disease through the induction of angiogenesis. However, the role of microRNA in angiogenesis induced by HCMV infection remains unclear. The present study was thus designed to explore the potential effect of miR-199a-5p on angiogenesis and to investigate the underlying mechanism in endothelial cells. We found that HCMV infection of endothelial cells (ECs) enhanced expression of miR-199a-5p and reduced the SIRT1 protein level at 24 h postinfection (hpi). Transfection with miR-199a-5p mimics significantly suppressed SIRT1 protein expression and promoted cellular migration and tube formation induced by HCMV infection, which could be reversed by transfection with an miR-199a-5p inhibitor. Furthermore, pretreatment with resveratrol depressed motility and tube formation of HCMV-infected ECs, which could be reversed by SIRT1 siRNA. Finally, overexpression of miR-199a-5p decreased the level of eNOS modulated by SIRT1, an effect repressed by transfection with an miR-199a-5p inhibitor. In summary, HCMV infection of endothelial cells upregulates miR-199a-5p expression and enhances cell migration and tube formation through downregulation of SIRT1/eNOS by miR-199a-5p.