Bisphenol AF (BPAF) has been widely used as a main alternative to bisphenol A (BPA), and previous in vitro studies have shown that BPAF has higher reproductive toxicity potentials than BPA. However, data on in vivo toxicity of BPAF is still limited. In this study, Sprague Dawley rats were exposed to BPAF (0, 50, and 100 mg/kg/day) during gestation to study toxicokinetics and reproductive toxicity in offspring. The results showed that plasma concentrations BPAF peaked within 6 h after birth, followed by a two-phase decay, with clearance rates of approximately 3.0 l/h and terminal half-life values ranging from 77 h to 114 h, suggesting fast absorption and high persistence of BPAF. At postnatal day 21 (PND21), BPAF was found to be bioaccumulated in reproductive organs (testes and ovaries) of the offspring, resulting in adverse effects on reproduction in both sexes. Lower anogenital distance, reduced relative testicular weight, dissolved interstitial cells, fewer primary spermatocytes, decreased testosterone levels, and increased luteinizing hormone levels were detected in male offspring. In female offspring, vacuolization in follicular antrum, fewer follicles, increased 17β-estradiol levels, and increased luteinizing hormone levels in female offspring were found. Gene expression of scavenger receptor class B type I (SR-B1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), sterol regulatory element-binding protein-1c (SREBP-1c), and several steroidogenic enzymes was significantly decreased in male offspring following maternal exposure to BPAF, suggesting that the decreases in testosterone levels is a result of inhibited cholesterol uptake, cholesterol de novo synthesis, and steroidogenesis. In addition, inhibition of pathways of phagosome and cell adhesion molecules might be the underlying molecular mechanism involved in BPAF-induced reproductive disorders in male offspring. This study provides the scientific basis for a comprehensive assessment of the safety of BPAF.