Purpose <p>Endolysosomal two-pore channels (TPCs) are non-selective cation channels that control the release of Ca²⁺ and Na⁺ from the endolysosomal lumen. TPCs also reportedly play a role in autophagy. Interestingly, autophagy regulates bone cell differentiation and function. This study aimed to provide an in-depth insight into TPC2’s action in the autophagy pathway to control osteoblast differentiation and function.</p> Methods <p>Primary human mesenchymal stem cells (hMSCs) and human osteoblast-like cells (Saos-2) were used to assess osteoblastogenesis and bone mineralization, respectively. MSCs were treated with different pharmacological TPC2 inhibitors including naringenin, tetrandrine, MT-8 and SG-094 during their differentiation process. Finally, formation of osteoblasts and in vitro bone mineralization were evaluated by alkaline phosphatase, alizarin red S and Von Kossa staining. Western blot analysis was performed to investigate the expression of autophagy-related molecules.</p> Results <p>The inhibition of TPC2 activity stimulates osteoblast differentiation from hMSCs and bone mineralization by Saos-2 cells. Interestingly, TPC2 inhibition reduces beclin-1 and LC3-II expression while that of the mammalian target of rapamycin (mTOR), the master regulator of autophagy, increases. Inhibition of mTOR activity by rapamycin reverses osteoblast differentiation induced by TPC2 inhibitor SG-094.</p> Conclusion <p>Inhibition of TPC2 channel activity increases osteoblast differentiation and bone mineralization in vitro and interferes with the completion of autophagy, upregulating phosphorylated mTOR.</p>