Stem cells have been a recent focus of regenerative medicine research. They originate from various tissues including human dental pulp containing human deciduous tooth dental pulp cells (hDPCs) that feature in the field of dentistry. However, only a very small number of stem cells are present in dental pulp tissue. It is known that 6-bromoindirubin-3'-oxime (BIO), a specific glycogen synthase kinase-3 (GSK-3) inhibitor, maintains the undifferentiated state of human and mouse embryonic stem cells. However, there are few reports that have applied BIO to hDPCs. Thus, we hypothesized that BIO may be necessary to maintain the undifferentiated state of hDPCs for clinical application. The aim of this study was to evaluate the effect of BIO on the undifferentiated state of hDPCs. hDPCs were extracted from the non-caries deciduous teeth of healthy children. BIO treatment at 0.5-1.5 μM slightly affected the proliferation of hDPCs, and 1-1.5 μM BIO induced the expression of Oct3/4 and Sox2 genes. Conversely, 0.5-1.5 μM BIO significantly reduced the gene expression of c-Myc as determined by real-time RT-PCR. Moreover, 1 μM BIO induced the expression of CD44 and CD90 as determined by flow cytometry. In summary, we found that 1 μM BIO was optimal for maintaining the undifferentiated state of hDPCs.