In this study, the effects of irisin, a hormone secreted by skeletal muscle, able to improve metabolic homeostasis and promote energy expenditure, on the β-cell functional mass were evaluated both in diabetic mice in vivo and in human pancreatic islets isolated from subjects with type 2 diabetes (T2D) ex vivo. Moreover, the molecular mechanisms underlying irisin action were investigated. C57Bl/6 mice (n=8) were made diabetic by a high-fat diet (HFD) and a single-dose of streptozotocin (STZ). 4 standard diet (SD)-fed mice were used as control. HFD/STZ mice were treated with 0.5 μg/g irisin (n=4) or vehicle (n=4) for 14 days. Fasting glycemia, insulinemia, body weight, glucose tolerance, and GSIS were assessed. Pancreatic islet architecture was evaluated by immunofluorescence analyses. Pancreatic islets isolated from T2D patients (n=14) and non-diabetic subjects (ND) (n=10), as well as INS-1E cells, were exposed to 100 nM irisin for different times. GSIS and insulin content were measured by ELISA assays; apoptosis was evaluated by the measurement of cytoplasmic oligosomes concentration and TUNEL assay; intracellular signaling proteins and calcium levels were evaluated by immunoblotting and fluorimetric assay, respectively. Irisin administration improved glycemic homeostasis, reduced body weight, and increased β-cell functional mass by stimulating β-cell proliferation and enhancing GSIS in vivo. Moreover, in islets from T2D subjects, characterized by functional and structural defects, irisin augmented insulin content and glucose-stimulated insulin secretion. Of note, irisin was able to activate CREB and AKT in INS-1E cells under physiological conditions, but not under glucotoxic conditions. However, while chronic exposure to excess glucose blunted both GSIS and glucose-evoked increase in cytoplasmic calcium levels, irisin restored insulin secretion through the mobilization of calcium stores from the endoplasmic reticulum. Thus, the mechanisms underlying the secretagogue effects of irisin in the type 2 diabetes islets are apparently independent of CREB and AKT activation but could involve direct effects on calcium delivery from ER stores and the exocytosis machinery of insulin granules. These findings highlight the role of irisin as a hormone that counteracts β-cell failure under dysmetabolic conditions in both rodents and humans and could be used to delay T2D onset and/or progression. Supported by PRIN 2020, NextGenerationEU (Age-it), Fondazione Cianciola.