Bisphenol A (BPA) is a well-known plasticizer that widely distributed in the aquatic environment. BPA has many adverse effects on reproduction. However, few studies have investigated the mechanism of BPA affecting reproduction from the perspective of lipid metabolism. Apolipoprotein A1 (ApoA1) is the major component of high-density lipoprotein (HDL), and plays critical roles in reverse cholesterol transport (RCT). In this study, in order to investigate the effect and molecular mechanism of BPA on testicular ApoA1 and the role of ApoA1 in BPA induced abnormal spermatogenesis, adult male rare minnow Gobiocypris rarus were exposed to 15μg/L of BPA for 1, 3 and 5 weeks. Results showed that BPA could significantly affect testicular ApoA1 mRNA and protein levels, testicular cholesterol levels, plasmatic sex hormone levels and the integrity of sperm head membrane. The main mechanism of BPA regulating ApoA1 expression is to alter Esr recruitment and CpG sites DNA methylation in ApoA1 promoter. The induced ApoA1 up-regulated high density lipoprotein cholesterol levels and enhanced RCT, and finally decreased the testicular free cholesterol levels. This is likely a key mechanism by which BPA induces sex hormone disorder and sperm head membrane damage. The present study reveals the mechanism by which BPA interferes with spermatogenesis from the perspective of cholesterol transport.