Chitosan, a natural biopolymer derived from chitin, is considered a promising scaffold material for bone tissue engineering. The ability of chitosan to promote the osteogenic differentiation of dental pulp stromal/stem cells (DPSCs) is unknown. We have evaluated the potential of chitosan to induce the osteogenic differentiation of macaque DPSCs in comparison with that of dexamethasone. DPSCs were cultured in mineralizing medium supplemented with 5 or 10 μg/ml chitosan or with 1 or 10 nM dexamethasone. The metabolic activity of DPSCs was measured by MTT assay. Their osteogenic differentiation was determined by the number of transcripts of RUNX2, alkaline phosphatase (ALP), and COL1A1 by using real-time polymerase chain reaction, by alizarin red staining for mineral deposition, and by the ALP activity released into the medium for their ability to support biomineralizaton. Addition of chitosan to the mineralizing medium significantly increased DPSCs metabolism after 7 and 14 days of culture (P ≤ 0.0001). Chitosan at 5 μg/ml also significantly enhanced RUNX2 and ALP mRNA but not COL1A1 mRNA; chitosan tended to increase the release of ALP hydrolytic enzyme activity into the medium during the first week. Dexamethasone upregulated the osteogenic markers tested. Mineral deposition was similar in the chitosan and dexamethasone groups and was not statistically different from that of the mineralizing control group. Thus, the potential of chitosan to stimulate DPSCs proliferation and early osteogenic differentiation is comparable with that of dexamethasone, but mineralization remains unaffected by chitosan treatment. In addition to its role as a three-dimensional scaffold for osteogenic cells in vivo, chitosan might also stimulate DPSCs proliferation and early osteogenic differentiation in vitro.