OBJECTIVE Hyperinsulinemia is commonly associated with obesity. Mice deficient in the adipose-derived hormone leptin (Lep(ob/ob)) develop hyperinsulinemia prior to onset of obesity and glucose intolerance. Whether the excess of circulating insulin is a major contributor to obesity and impaired glucose homeostasis in Lep(ob/ob) mice is unclear. It has been reported previously that diet-induced obesity in mice can be prevented by reducing insulin gene dosage. In the present study, we examined the effects of genetic insulin reduction in Lep(ob/ob) mice on circulating insulin, body composition, and glucose homeostasis. METHODS Leptin expressing (Lep(wt/wt)) mice lacking 3 insulin alleles were crossed with Lep(ob/ob) mice to generate Lep(ob/ob) and Lep(wt/wt) littermates lacking 1 (Ins1(+/+);Ins2(+/-)), 2 (Ins1(+/+);Ins2(-/-)) or 3 (Ins1(+/-);Ins2(-/-)) insulin alleles. Animals were assessed for body weight gain, body composition, glucose homeostasis, and islet morphology. RESULTS We found that in young Lep(ob/ob) mice, loss of 2 or 3 insulin alleles reduced plasma insulin levels by 75-95% and attenuated body weight gain by 50-90% compared to Ins1(+/+);Ins2(+/-);Lep(ob/ob) mice. This corresponded with ∼30% and ∼50% reduced total body fat in Ins1(+/+);Ins2(-/-);Lep(ob/ob) and Ins1(+/-);Ins2(-/-);Lep(ob/ob) mice, respectively. Loss of 2 or 3 insulin alleles in young Lep(ob/ob) mice resulted in onset of fasting hyperglycemia by 4 weeks of age, exacerbated glucose intolerance, and abnormal islet morphology. In contrast, loss of 1,2 or 3 insulin alleles in Lep(wt/wt) mice did not significantly alter plasma insulin levels, body weight, fat mass, fasting glycemia, or glucose tolerance. CONCLUSION Taken together, our findings indicate that hyperinsulinemia is required for excess adiposity in Lep(ob/ob) mice and sufficient insulin production is necessary to maintain euglycemia in the absence of leptin.