Diabetes is a complex disease that impacts more than 500 million people across the world. Many of these individuals will develop diabetic neuropathy as a comorbidity, which is historically treated with exogenous opioids like morphine, oxycodone, or tramadol. Although these opioids are effective analgesics, growing evidence indicates that they may directly impact the endocrine pancreas function in patients. One common feature of these exogenous opioid ligands is their preference for the mu opioid receptor (MOPR), so we aimed to determine if endogenous MOPRs directly regulate pancreatic islet metabolism and hormone secretion. We show that pharmacological antagonism of MOPRs enhances glucagon secretion, but not insulin secretion, from human islets under high glucose conditions. This increased secretion is accompanied by increased cAMP signaling. mRNA expression of MOPRs is robust in non-diabetic human islets, but downregulated in islets from T2D donors, suggesting a link between metabolism and MOPR expression. Conditional genetic knockout of MOPRs in murine α-cells increases glucagon secretion under high glucose conditions without increasing glucagon content. Consistent with downregulation of MOPRs during metabolic disease, conditional MOPR knockout mice treated with a high fat diet show impaired glucose tolerance, increased glucagon secretion, increased insulin content, and increased islet size. Together, these results demonstrate a direct mechanism of action for endogenous opioid regulation of endocrine pancreas.