Dental stem cells, especially dental follicle cells (DFCs) as precursor cells for the periodontium have interesting prospects for regenerative dentistry. During periodontitis, butyrate as a bacterial metabolite and inflammatory agent is often found in millimolar concentrations in periodontal pockets. This study evaluates the effects of butyrate on the proliferation and osteogenic differentiation of DFCs. We assessed cell viability/proliferation (BCA assay) and osteogenic differentiation (ALP activity, alizarin staining and RT PCR) of DFCs in vitro after butyrate supplementation. Butyrate concentrations of 20 mM or higher are toxic for DFCs. At a non-toxic concentration, butyrate promotes the expression of alkaline phosphatase and collagen type-1 but inhibits the formation of calcified nodules and the induction of RUNX2 and osteocalcin under osteogenic differentiation conditions. In conclusion, DFCs are resistant to physiological high concentrations of butyrate. Butyrate facilitates the osteogenic differentiation of DFCs in early stages but inhibits calcification at later stages of the differentiation process.