Mitochondrial dysfunction is associated with beta-cell failure and insulin resistance in diabetes. Humanin is an endogenous cytoprotective peptide. In the current study, we aimed to define the effects of Humanin on mitochondrial biogenesis in pancreatic beta-cells. Our findings demonstrated that Humanin treatment significantly increased the expression of PGC-1 alpha and its downstream target genes NRF1 and TFAM in MIN6 beta-cells. Notably, Humanin treatment significantly promoted mitochondrial biogenesis by increasing mitochondria! mass, elevating mtDNA/nDNA ratio, and stimulating the expression of cytochrome B, which were suppressed by the specific AMPK inhibitor compound C. Indeed, Humanin treatment caused the phosphorylation of AMPK, which was involved in the induction of PGC-1 alpha, NRF1, and TFAM by Humanin. Importantly, our findings indicate that Humanin treatment led to a possible functional gain of the mitochondria by increasing ATP levels and respiratory rate. Our findings provided a new insight into the molecular mechanisms of action by which Humanin improves pancreatic beta-cell function via enhanced mitochondrial mass and performance. (C) 2018 Published by Elsevier Inc.