Although autophagy is critical for pancreatic beta-cell function, the role and mechanism of mitophagy in beta-cells are unclear. We studied the role of lysosomal Ca2+ in TFEB activation by mitochondrial or metabolic stress and that of TFEB-mediated mitophagy in beta-cell function. Mitochondrial or metabolic stress induced mitophagy through lysosomal Ca2+ release, increased cytosolic Ca2+ and TFEB activation. Lysosomal Ca2+ replenishment by ER- > lysosome Ca2+ refilling was essential for mitophagy. beta-cell-specific Tfeb knockout (Tfeb(Delta beta-cell)) abrogated high-fat diet (HFD)-induced mitophagy, accompanied by increased ROS and reduced mitochondrial cytochrome c oxidase activity or O-2 consumption. Tfeb(Delta beta-cell) mice showed aggravation of HFD-induced glucose intolerance and impaired insulin release. Metabolic or mitochondrial stress induced TFEB-dependent expression of mitophagy receptors including Ndp52 and Optn, contributing to the increased mitophagy. These results suggest crucial roles of lysosomal Ca2+ release coupled with ER- > lysosome Ca2+ refilling and TFEB activation in mitophagy and maintenance of pancreatic beta-cell function during metabolic stress. Autophagy is important for pancreatic beta-cell function, however, the role of mitophagy and mechanism for mitophagy in beta-cells are unclear. Here the authors report that in stressed beta-cells, lysosomal Ca2+ release promotes mitophagy via activation of the transcription factor EB (TFEB) and loss of beta-cell TFEB aggravates glucose intolerance during high-fat diet.