To investigate the effects of autophagy on osteogenic differentiation of stem cells from the apical papilla (SCAPs) in the presence of tumor necrosis factor-α (TNF-α) stimulation in vitro.SCAPs treated with TNF-α (0, 5, and 10 ng/mL) with or without 5 mmol/L 3-MA were examined for the expression of autophagy marker LC3-Ⅱ using Western blotting. The cells were transfected with GFP-LC3 plasmid and fluorescence microscopy was used for quantitative analysis of intracellular GFP-LC3; AO staining was used to detect the acidic vesicles in the cells. The cell viability was assessed with CCK-8 assays and the cell apoptosis rate was analyzed using flow cytometry. The cells treated with TNF-α or with TNF-α and 3-MA were cultured in osteogenic differentiation medium for 3 to 14 days, and real- time PCR was used to detect the mRNA expressions of osteogenesis-related genes (ALP, BSP, and OCN) for evaluating the cell differentiation.TNF-α induced activation of autophagy in cultured SCAPs. Pharmacological inhibition of TNF-α-induced autophagy by 3-MA significantly decreased the cell viability and increased the apoptosis rate of SCAPs (P < 0.05). Compared with the cells treated with TNF-α alone, the cells treated with both TNF-α and 3-MA exhibited decreased expressions of the ALP and BSP mRNA on days 3, 7 and 14 during osteogenic induction (P < 0.05) and decreased expression of OCN mRNA on days 3 and 7 during the induction (P < 0.05).Autophagy may play an important role during the osteogenic differentiation of SCAPs in the presence of TNF-α stimulation.