Pterostilbene (Pt) is a potentially beneficial plant phenol. In contrast to many other natural compounds (including the more celebrated resveratrol), Pt concentrations producing significant effects in vitro can also be reached with relative ease in vivo. Here we focus on some of the mechanisms underlying its activity, those involved in the activation of transcription factor EB (TFEB). A set of processes leading to this outcome starts with the generation of ROS, attributed to the interaction of Pt with complex I of the mitochondrial respiratory chain, and spreads to involve Ca2+ mobilization from the ER/mitochondria pool, activation of CREB and AMPK, and inhibition of mTORC1. TFEB migration to the nucleus results in the upregulation of autophagy and lysosomal and mitochondrial biogenesis. Cells exposed to several μM levels of Pt experience a mitochondrial crisis, an indication for using low doses in therapeutic or nutraceutical applications. Pt afforded significant functional improvements in a zebrafish embryo model of ColVI-related myopathy, a pathology which also involves defective autophagy. Furthermore, long-term supplementation with Pt reduced body weight gain and increased transcription levels of Ppargc1a and Tfeb in a mouse model of diet-induced obesity. These in vivo findings strengthen the in vitro observations and highlight the therapeutic potential of this natural compound.