GPR39 is a G protein-coupled receptor expressed in liver, gastrointestinal tract, adipose tissue, and pancreas. We have recently shown that young GPR39^−/− mice have normal body weight, food intake, and fasting glucose and insulin levels. In this study, we examined the role of GPR39 in aging and diet-induced obese mice. Body weight and food intake were similar in wild-type and GPR39^−/− mice as they aged from 12 to 52 wk or when fed a low-fat/high-sucrose or high-fat/high-sucrose diet. Fifty-two-week-old GPR39^−/− mice showed a trend toward decreased insulin levels after oral glucose challenge. When fed either a low-fat/high-sucrose or high-fat/high-sucrose diet, GPR39^−/− mice had increased fed glucose levels and showed decreased serum insulin levels during an oral glucose tolerance test in the face of unchanged insulin tolerance. Pancreas morphology and glucose-stimulated insulin secretion in isolated islets from wild-type and GPR39^−/− mice were comparable, suggesting that GPR39 is not required for pancreas development or ex vivo insulin secretion. Small interfering RNA-mediated knockdown of GPR39 in clonal NIT-1 β-cells revealed that GPR39 regulates the expression of insulin receptor substrate-2 and pancreatic and duodenal homeobox-1 in a cell-autonomous manner; insulin receptor substrate-2 mRNA was also significantly decreased in the pancreas of GPR39^−/− mice. Taken together, our data indicate that GPR39 is required for the increased insulin secretion in vivo under conditions of increased demand, i.e. on development of age-dependent and diet-induced insulin resistance. Thus, GPR39 agonists may have potential for the treatment of type 2 diabetes.