Latrunculin depolymerizes and jasplakinolide polymerizes β-cell actin microfilaments. Both increase insulin secretion when Ca²⁺ enters β-cells during depolarization by glucose, sulfonylureas or potassium. Mouse islets were held hyperpolarized with diazoxide, and stimulated with acetylcholine to test the role of microfilaments in insulin secretion triggered by intracellular Ca²⁺ mobilization and store-operated Ca²⁺ entry (SOCE). Jasplakinolide slightly attenuated Ca²⁺ mobilization and did not affect SOCE, but consistently inhibited the attending insulin secretion. Latrunculin did not affect Ca²⁺ changes induced by acetylcholine, but consistently increased insulin secretion, its effect being larger in response to Ca²⁺ entry than to Ca²⁺ mobilization. Microfilaments have thus a distinct impact on exocytosis of insulin granules depending on the source of triggering Ca²⁺.