Introduction Dental mesenchymal stem cells (dMSCs) may differentiate into odontoblast-like cells and form mineralized nodules. In the current study, we investigated the effects of senescence on odontogenic differentiation of dMSCs. Methods dMSCs were serially subcultured until senescence. Telomere lengths and telomerase activities were determined by quantitative PCR. Expression of genes involved in cell proliferation and differentiation, e.g., Bmi-1, p16INK4A, osteocalcin (OC), dentin sialoprotein (DSP), bone sialoprotein (BSP), and dentin matrix protein-1 (DMP-1) were assayed by Western blotting and quantitative reverse transcription PCR. Exogenous Bmi-1 was expressed in dMSC using retroviral vectors. Odontogenic differentiation was assayed by alkaline phosphatase (ALP) activity. Results Subculture-induced replicative senescence of dMSCs led to reduced expression of Bmi-1, OC, DSPP, and BSP compared with rapidly proliferating cells, while p16INK4A level increased. The cells exhibited progressive loss of telomeric DNA during subculture, presumably due to lack of telomerase activity. Bmi-1 transduction did not affect proliferation of cells, but enhanced the expression of OC and DSPP in the late passage cultures. Bmi-1-transduced cells also demonstrated enhanced ALP activity and mineralized nodule formation. Conclusions These results indicate that dMSCs lose their odontogenic differentiation potential during senescence, in part, by reduced Bmi-1 expression.