Human dental pulp contains a rapidly proliferative subpopulation of precursor cells termed dental pulp stem cells (DPSCs) that show self‐renewal and multilineage differentiation, including neurogenic, chondrogenic, osteogenic and adipogenic. We previously reported that tomuor necrosis factor‐α (TNF‐α) (10 ng/mL) triggered osteogenic differentiation of human DPSCs via the nuclear factor‐κB (NF‐κB) signaling pathway. While previous studies showed that cells treated with TNF‐α at higher concentrations showed decreased osteogenic differentiation capability. In this study we analyze the function of TNF‐α (100 ng/mL) on osteogenic differentiation of human DPSCs for the first time and identify the underlying molecule mechanisms. Our data revealed that TNF‐α with higher concentration significantly reduced mineralization and the expression of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP) and runt‐related transcription factor 2 (RUNX2). Further, we revealed that TNF‐α could suppress the osteogenic differentiation of DPSCs via increasing the expression of RAC1, which could activate the Wnt/β‐catenin signaling pathway and liberate β‐catenin to translocate into the nucleus. Genetic silencing of RAC1 expression using siRNA restored osteogenic differentiation of DPSCs. Our findings may provide a potential approach to bone regeneration in inflammatory microenvironments.