In several rodent strains such as the New Zealand Obese (NZO) mouse, the incidence of obesity-associated diabetes mellitus is much higher in males than in females. In the present study, we investigated the effects of ovariectomy on glucose homeostasis in female NZO mice in order to elucidate the mechanism of their diabetes resistance. NZO females were ovariectomized at the age of 4 weeks, received a high-fat diet and body weight, body fat, glucose and insulin tolerance were investigated in comparison to sham-operated mice. In a second experiment, operated mice were fed a carbohydrate-free diet up to the age of 19 weeks before they received the high-fat diet. In comparison with a sham-operated control group, ovariectomized female NZO mice exhibited similar body weights, a reduced glucose tolerance, developed significantly higher blood glucose levels, lost insulin producing β-cells, which finally resulted in a diabetes prevalence of 73% at the age of 16 weeks vs. 25% in controls. Similar to male NZO mice, ovariectomized females presented a more severe insulin resistance in the insulin tolerance test than sham-operated controls. Furthermore, the more severe insulin resistance in ovariectomized mice preceded the development of diabetes and pancreatic insulin depletion that was caused by a dietary regimen of carbohydrate restriction and subsequent re-exposure. In summary our data demonstrate that estrogen protects NZO females from β-cell loss and obesity-associated diabetes mellitus. This effect is due to a reduced insulin resistance and possibly also to a reduced sensitivity of β-cells to glucolipotoxic conditions.