Natural and synthetic steroid hormones have been detected in swine farm wastewaters (SFWs) and receiving waters. However, little is known on their potential endocrine disrupting effects on fish population in receiving rivers. Here we investigated the concentrations of androgens and estrogens in the SFWs and the receiving waters. In addition, the endocrine disrupting effects of SFWs on wild western mosquitofish (Gambusia affinis) were also analyzed by assessing the sex ratio, secondary sex characteristics and transcriptional expression of genes related to the endocrine system. Chemical analysis showed the maximum concentration of total androgens reached 1375.7 ng L−1 in the SFWs and 1020.8 ng L−1 in receiving river waters. A total of 3552 adult G. affinis were collected from the Lianhe River (Guangdong, China), which was heavily impacted by SFWs, before and after the Ban of Swine Farming (BSF) in the river basin. The results showed the presence of male-biased G. affinis populations and masculinized anal fins and hemal spines of females at all contaminated sites prior to BSF. In addition, the transcription of vitellogenin (vtg) was inhibited by SFWs by 99.9% in the females from one sampling site. In contrast, the adverse effects on the females were significantly decreased after BSF. Redundant and path analyses showed that skewed sex ratios of fish populations, masculinized secondary sex characteristics of females and the decrease of vtg transcripts of females were well correlated with the concentrations of androgens in the receiving waters. The findings from this study suggest that SFW can cause masculinization effects of wild G. affinis populations. Keywords: Swine farm wastewater, Hormones, Androgens, Gambusia affinis, Masculinization