OBJECTIVE: Dynorphin, an endogenous opioid peptide predominantly expressed in the central nervous system and involved in stress response, pain, and addiction, has intrigued researchers due to its expression in pancreatic β-cells. In this study, we aimed to characterize dynorphin expression in mouse and human islets and explore the mechanisms regulating its expression. METHODS: We used primary mouse and human islets with unbiased published datasets to examine how glucose and other nutrients regulate dynorphin expression and secretion in islets. RESULTS: The prodynorphin gene is significantly upregulated in β-cells under hyperglycemic conditions. In vitro studies revealed that increased glucose concentrations correlate with increased dynorphin expression, indicating a critical interplay involving Ca CONCLUSIONS: This study demonstrates that the opioid peptide prodynorphin is expressed in mouse and human β-cells. Prodynorphin levels are regulated in parallel with insulin in response to glucose, palmitate, and amino acids. Our findings elucidate the signaling pathways involved, with CamKII playing a key role in regulating prodynorphin levels in β-cells. Finally, our findings are the first to demonstrate active dynorphin secretion from mouse and human islets in response to glucose.