Intrauterine environment may influence the health of postnatal offspring. There have been many studies on the effects of maternal high fat diet (HFD) on diabetes and glucose metabolism in offspring. Here, we investigated the effects in male and female offspring. C57/BL6J mice were bred and fed either control diet (CD) or HFD from conception to weaning, and offspring were fed CD or HFD from 6 to 20 weeks. At 20 weeks, maternal HFD induced glucose intolerance and insulin resistance in offspring. Additionally, liver triacylglycerol content, adipose tissue mass and inflammation increased in maternal HFD. In contrast, extending previous observations, insulin secretion at glucose tolerance test, islet area, insulin content and PDX-1 mRNA levels in isolated islets were lower in maternal HFD in male, while they were higher in female. Oxidative stress in islets increased in maternal HFD in male, while there were no differences in female. Plasma estradiol levels were lower in male than in female, and decreased in offspring fed with HFD, and also decreased by maternal HFD, suggesting that females may be protected from insulin deficiency by inhibiting oxidative stress. In conclusion, maternal HFD induced insulin resistance and deterioration of pancreatic β cell function with marked gender differences in adult offspring, accompanied by adipose tissue inflammation and liver steatosis. Additionally, our results demonstrate that potential mechanisms underlying gender differences in pancreatic β cell function may be partially related to increases in oxidative stress in male islets and decreased plasma estradiol levels in male.