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AIM To investigate the role of autophagy in MTA-induced odontoblastic differentiation of human dental pulp cells (HDPCs). METHODOLOGY In MTA-treated HDPCs, odontoblastic differentiation was assessed based on expression levels of dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP1), alkaline phosphatase activity (ALP) activity by ALP staining and the formation of mineralized nodule by Alizarin red S staining. Expression of microtubule-associated protein 1A/1B-light chain3 (LC3), adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling molecules and autophagy-related genes, were analyzed by Western blot analysis and Acridine orange staining was used to detect autophagic lysosome. For in vivo experiments, tooth cavity preparation models on rat molars was established and the expression of proteins related odontogenesis and autophagy markers were observed by Immunohistochemistry and Western blot analysis. Kruskal-Wallis with Dunn's multiple comparison was used for statistical analysis. RESULTS MTA promoted odontoblastic differentiation of HDPCs, accompanied by autophagy induction, including formation of autophagic lysosome and cleavage of LC3 to LC3II (P < 0.05). Conversely, inhibition of autophagy through 3MA significantly attenuated the expression level of DSPP (P < 0.05) and DMP1 (P < 0.05) as well as formation of mineralized nodules (P < 0.05), indicating the functional significance of autophagy in MTA-induced odontoblastic differentiation. Also, MTA increased the activity of AMPK (P < 0.01), whereas inhibition of AMPK by compound C downregulated DSPP (P < 0.01) and DMP1 (P < 0.05), but increased the phosphorylation of mTOR (P < 0.05), p70S6 (P < 0.01) and Unc-51-like kinases 1 (ULK1)(ser757) (P < 0.01), explaining the involvement of AMPK pathway in MTA-induced odontoblast differentiation. In vivo study, MTA treatment after tooth cavity preparation on rat molars upregulated DMP-1 and DSPP as well as autophagy-related proteins LC3II and p62, and enhanced the phosphorylation of AMPK. CONCLUSION MTA induced odontoblastic differentiation and mineralization by modulating autophagy with AMPK activation in HDPCs. Autophagy regulation is a new insight on regenerative endodontic therapy using MTA treatment.