Mesenchymal stem cells (MSCs) have both multi-lineage differentiation potential and immunosuppressive properties, making them ideal candidates for regenerative medicine. However, their immunosuppressive properties potentially increase the risk of cancer progression and opportunistic infections. In this study, MSCs isolated from human umbilical cord blood (UCMSCs) and adult bone marrow (BMMSCs) were infected with human cytomegalovirus (HCMV). Cytopathic changes were observed 10 days post infection. PCR products amplified from genomic DNA and cDNA were used to confirm the HCMV infection of the UCMSCs and BMMSCs. Real-time PCR was conducted to quantify the expression of immunomodulatory molecules, including cytokines, chemokines, growth factors, adhesion molecules and cancer-related genes. Our results indicate high upregulation of the majority of these molecules, including many growth factors, tumor necrosis factor alpha, interleukin-8, interleukin-6 and interferon gamma. Adhesion molecules (VCAM-1, TCAM-1 and selectin-E) were downregulated in the infected UCMSCs and BMMSCs. Antibody chip array evaluation of cell culture media indicated that the growth factor secretion by UCMSCs and BMMSCs was greatly influenced (p < 0.001) by HCMV. The stimulation of MSCs with HCMV led to the activation of downstream signaling pathways, including pSTAT3 and Wnt2. Our results show that HCMV can significantly alter the functions of both UCMSCs and BMMSCs, although not in the same way or to the same extent. In both cases, there was an increase in the expression of proangiogenic factors in the microenvironment following HMCV infection. The discrepancy between the two cell types may be explained by their different developmental origin, although further analysis is necessary. Future studies should decipher the underlying mechanism by which HCMV controls MSCs, which may lead to the development of new therapeutic treatments.