PURPOSE: We previously demonstrated that hyperosmotic stress, which acts as mechanical stress, induces autophagy of tubular epithelial cells. This study aims to elucidate the molecular mechanisms of hyperosmolarity-induced autophagy. The research question addresses how hyperosmotic stress activates autophagy through transcription factor EB (TFEB) and Ca METHODS: NRK-52E normal rat kidney cells were subjected to hyperosmotic stress using mannitol-containing medium. Fluorescence microscopy was utilized to observe TFEB nuclear translocation, a crucial event in autophagy regulation. An intracellular Ca RESULTS: Mannitol-induced hyperosmotic stress promoted the nuclear translocation of TFEB, which was completely abolished by treatment with BAPTA-AM. Inhibition of calcineurin suppressed TFEB nuclear translocation under hyperosmolarity, indicating that a signaling pathway governed by intracellular Ca CONCLUSIONS: Hyperosmotic stress promotes TFEB nuclear localization, and TRPML1-induced activation of calcineurin is involved in the mechanism of hyperosmolarity-induced autophagy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-024-00839-6.