Xanthohumol (XN) is a prenylated chalcone, which has been shown to possess a broad range of potential cancer preventive and additional biological activities. In the present study, we have determined the subchronic 4-wk toxicity of XN and monitored its influence on fertility and development of offspring in two fertility studies. Four-week-old female Sprague Dawley (SD) rats were treated with 0.5% XN in the diet or with 1000 mg XN/kg body weight (b.w.) per day by gavage for 28 days. No remarkable treatment-related changes in general appearance and b.w. occurred during the study. After autopsy, liver, kidney, lung, heart, stomach, and spleen were examined macroscopically and histopathologically. Relative liver weights of animals in both treatment groups were significantly reduced by 30–40% in comparison with the control group, indicating weak hepatotoxicity. Also, mammary glands of treated rats appeared less developed compared to the controls. Consequently, we investigated the influence of XN on rat reproduction. In two fertility studies, XN (100 mg/kg b.w. per day), given either for 4 wk prior to or during mating, gestation, and nursing, did not cause any adverse effects on female reproduction and the development of offspring. Noteworthy, treatment of male rats prior to mating significantly (p = 0.027) increased the sex ratio of male to female offspring. Overall, lifelong treatment at a daily dose of 100 mg/kg b.w. in a two-generation study did not affect the development of SD rats.