Because of the central role of the transcription factor nuclear factor (NF)-κB in cell survival and proliferation in many kinds of cancer cells, NF-κB inhibitors may have a potential role in cancer therapy. Currently, many NF-κB inhibitors are used for immunosuppression to treat hematologic malignancy patients after stem cell transplantation (SCT). Human cytomegalovirus (HCMV) infection is one of the most common complications following SCT. Some workers have reported that HCMV infection has a close relationship to NF-κB activation; however, the specific effects of NF-κB inhibitors, such as pyrrolidinedithiocarbamate (PDTC), on infection with and activation of CMV in mesenchymal stem cells (MSCs) remain unknown. In our study, we isolated MSCs from the bone marrows of healthy human donors for infection with 1 tcid(50) of HCMV with or without 1 μmol/L PDTC. After 48 hours of culture in dmem supplemented with 10% (volume per volume) fetal calf serum, we tested MSCs using reverse transcription-polymerase chain reaction (RT-PCR) assays to detect messenger RNA (mRNA) expression of HCMV immediate early (IE) gene and the GAPDH gene. Flow cytometry was used to detect HCMV pp65 antigen-positive cells and transmission electron microscopy (TEM) for intra cellular HCMV particles. We observed that the shape of the MSCs changed in response to infection by 1 TCID(50) of HCMV. MSCs infected by 1 TCID(50) of HCMV in combination with 1 μmol/L of PDTC changed their shapes more profoundly; almost all cells went from a thin elongated profile to a round, thick ball. In contrast, the shape of cells treated with PDTC alone or the HCMV mock-infected elements did not change. The RT-PCR assay showed that there was a bright band corresponding to HCMV IE mRNA in MSCs infected with 1 TCID(50) of HCMV in combination with 1 μmol/L of PDTC, as compared with cells infected by only 1 TCID(50) of HCMV. The HCMV mock-infected MSCs did not express HCMV IE mRNA. Using flow cytometry, we detected more HCMV pp65 antigen-positive cells among MSCs infected with 1 TCID(50) of HCMV in combination with 1 μmol/L of PDTC. HCMV particles were observed by TEM in the nucleus and cytoplasm of MSCs infected with HCMV. There were more HCMV particles in cells infected by HCMV in combination with PDTC. In conclusion, NF-κB activation may affect HCMV infection efficiency of MSCs. An NF-κB inhibitor increased the infection by activation of HCMV in MSCs, thus we should pay close attention to HCMV infection when we prescribe an NF-κB inhibitor in clinical settings.