The free fatty acid (FFA) receptor GPR40, expressed by pancreatic β-cells, may be responsible for insulin release following β₃ adrenoceptor (Adrb3) activation. To test this hypothesis, we first studied the effects of Adrb3 agonists SR58611A and CL316,243 in GPR40 knockout (GPR40^−/−) mice. Both drugs increased blood FFA levels in wild-type (GPR40^+/+) and GPR40^−/− mice, indicating that lipolysis is not GPR40-dependent. However, the magnitude of the insulin response after agonist treatment was decreased by ∼50% in GPR40^−/− mice. Analysis of the time-course revealed that the change in FFAs (5–10min post-treatment) in response to SR58611A preceded insulin secretion (10–15min post-treatment). While reduced by agonist treatment, glucose levels in GPR40^−/− mice remained significantly higher than in GPR40^+/+ mice. Energy expenditure, food intake, or body weight was not affected in GPR40^−/− mice, whereas SR58611A increased energy metabolism. Furthermore, CL316,243 did not potentiate glucose-stimulated insulin secretion in isolated mouse islets or activate a cAMP reporter in transgenic mice. Our data indicate that insulin secretion, a secondary event following stimulation of Adrb3 receptors, is partially mediated by GPR40 and suggest that GPR40 is integral to the anti-diabetes effects of Adrb3 agonists.