Human dental pulp stem cells become an alternative source of stem cells that play an important role in tissue engineering and cell-based therapies that will help the regeneration of impaired tissue in elderly, regarding to the easy access and the potential to renew and differentiate into many cell types. Cell expansion in vitro is necessary to amplify the small amount of cells that can be isolated before therapeutic use of DPSC. However, long-term cultivation leads to the alteration in morphology, proliferation and differentiation potential of DPSCs. Moreover, the accumulation of mutation during cell expansion can bring the risk of malignant transformation. Epigenetic mechanism including DNA methylation was found taking part in the regulatory processes in development. The global loss of methylation level relates with many events including tumor progression. Hypomethylation of LINE-1 is associated with many types of cancer and occurs during early event in carcinogenesis. In this study, we examined and compared the methylation levels of LINE-1 between the early and late passages of DPSCs using combined bisulfite restriction analysis. DPSCs showed the morphological change and lost methylation level of LINE-1 during expansion. DPSCs in late passage have lower level of LINE-1 methylation than the early passage but with no statistical significant difference. Further study about epigenetic and malignant transformation of DPSCs is still recommended and required for the secure application of these cells.