Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, plays an important role in regulating glucose metabolism. The aim of this study was to investigate the effect of DHA on insulin secretion and the underlying ion channel mechanism in rat pancreatic β-cells. The insulin secretion results illustrated that DHA promoted insulin secretion in a glucose-dependent manner. Calcium-imaging analysis indicated that DHA elevated intracellular Ca²⁺ concentration. Using the patch-clamp technique, we found that DHA prolonged the action potential duration (APD) and significantly inhibited voltage-dependent K⁺ (K_V) channels, but did not act directly on voltage-gated Ca²⁺ channels. Furthermore, our data demonstrate that the insulinotropic effect of DHA was mediated by G protein-coupled receptor 40 (GPR40) as well as the adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP)/phospholipase-C (PLC) signaling pathway. Together, these findings illustrate that K_V channels play a vital role in DHA-augmented insulin secretion through a mechanism whereby DHA blocks K_V channels via GPR40 and the AC/cAMP/PLC signaling pathway in rat pancreatic β-cells.