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Cytopiloyne was identified as a novel polyacetylenic compound. However, its antidiabetic properties are poorly understood. The aim of the present study was to investigate the anti‐diabetic effect and mode of action of cytopiloyne on type 2 diabetes (T2D). We first evaluated the therapeutic effect of cytopiloyne on T2D in db/db mice. We found that one dose of cytopiloyne reduced postprandial glucose levels while increasing blood insulin levels. Accordingly, long‐term treatment with cytopiloyne reduced postprandial blood glucose levels, increased blood insulin, improved glucose tolerance, suppressed the level of glycosylated hemoglobin A_1c (HbA_1c), and protected pancreatic islets in db/db mice. Next, we studied the anti‐diabetic mechanism of action of cytopiloyne. We showed that cytopiloyne failed to decrease blood glucose in streptozocin‐ (STZ‐)treated mice whose β cells were already destroyed. Additionally …