Scope: β-Carotene (BC), a substitute for vitamin A, is widely used for its benefits. The present study investigates whether in-utero BC administration can alter lipid and glucose homoeostasis in offspring. Methods and results: Pregnant mice are supplemented with BC (1 mg kg −1 weight) by oral gavage once every 3 days, for a total of six doses. Increased visceral fat may be caused by up-regulated PPARγ (peroxisome proliferator-activated receptor gamma) and RXRα/β (retinoid X receptors) in liver and adipose tissue, and glucose intolerance is observed in F1 adult females prenatally supplemented with BC, while F1 males do not exhibit these symptoms. In females, increased serum leptin, resistin, and IL-6 and reduced adiponectin, caused by visceral obesity, may result in downregulated insulin receptor signaling in muscle and further account for glucose intolerance. Increased pancreatic β-cell mass might compensate for the downregulated insulin gene (ins2). Increased glucagon and α-cell mass, accompanied by upregulated glucagon gene (gcg), might also be risk factors for the development of diabetes. Conclusions: Maternal supplementation with BC disturbs lipid metabolism and induces glucose intolerance in F1 female mice, suggesting that BC supplementation during pregnancy should be used with caution.