Hematopoietic stem cells recipients remain susceptible to opportunistic viral infections including herpes simplex virus type‐1 (HSV‐1). The purpose of this investigation was to analyze susceptibility of human mesenchymal stem cells (hMSCs) to HSV‐1 infection and identify the major entry receptor. Productive virus infection in hMSCs was confirmed by replication and plaque formation assays using a syncytial HSV‐1 KOS (804) virus. To examine the significance of entry receptors, RT‐PCR and antibody‐blocking assays were performed. RT‐PCR data showed the expression of gD receptors: nectin‐1, 3‐O sulfotransferase‐3 (3‐OST‐3), and HVEM. Antibody‐blocking assay together with heparinase treatment suggested an important role for HS and 3‐O‐sulfated heparan sulfate (3‐OS HS), but not nectin‐1 or HVEM, in mediating HSV‐1 entry and spread in hMSCs. Taken together, our results provide strong evidence demonstrating that HSV‐1 is capable of infecting hMSCs and HS and 3‐OS HS serve as its entry receptors during this process.