BACKGROUND AND PURPOSE: Tooth eruption is a complicated process regulated by the dental follicles (DF). Our recent study discovered that tooth eruption was inhibited upon injection of bleomycin into DF. However, the mechanisms were unknown. EXPERIMENTAL APPROACH: Human dental follicle cells (hDFCs) were treated by bleomycin or exogenous TGF-β1 or transfected by plasmids loading SMAD7 or shRNA targeting SMAD7, followed by osteogenesis induction assay and signalling analysis. Human fresh DF tissues and Wistar rats were used to further confirm bleomycin function. KEY RESULTS: Bleomycin decreased expression of RUNX2 and osteogenic genes in hDFCs, reducing osteogenic capacity. TGF-β1 expression was up-regulated in bleomycin-treated hDFCs. The effects of exogenous TGF-β1 were similar to those of bleomycin in hDFCs. Additionally, compared to SMAD2/3, SMAD7 expression increased more in bleomycin- or TGF-β1-treated hDFCs. Overexpression of SMAD7 likewise significantly decreased RUNX2 expression and osteogenic capacity of hDFCs. Knockdown of SMAD7 markedly attenuated the inhibitory effects of bleomycin and TGF-β1 on osteogenic capacity and RUNX2 expression of hDFCs. Most importantly, changes in TGF-β1, SMAD7, and RUNX2 expressions were similar in the DF of rats and humans treated with bleomycin. CONCLUSION AND IMPLICATIONS: SMAD7 was a negative regulator of osteogenic differentiation in DFCs through suppressing RUNX2 expression. Bleomycin or TGF-β1 inhibited osteogenic differentiation of DFCs via a TGF-β1/SMAD7/RUNX2 pathway. Our findings might be beneficial for enhancing the osteogenic activity of DFCs or inhibiting the eruption of undesirable teeth.