Propylparaben (PrP) is a widely used preservative that is constantly detected in aquatic environments and poses a potential threat to aquatic ecosystems. In the present work, adult male mosquitofish were acutely (4d) and chronically (32d) exposed to environmentally and humanly realistic concentrations of PrP (0, 0.15, 6.00 and 240 μg/L), aimed to investigate the toxic effects, endocrine disruption and possible mechanisms of PrP. Histological analysis showed time- and dose-dependent manners in the morphological injuries of brain, liver and testes. Histopathological alterations in the liver were found in 4d and severe damage was identified in 32d, including hepatic sinus dilatation, cytoplasmic vacuolation, cytolysis and nuclear aggregation. Tissue impairments in the brain and testes were detected in 32d; cell cavitation, cytomorphosis and blurred cell boundaries appeared in the brain, while the testes lesions contained spermatogenic cell lesion, decreased mature seminal vesicle, sperm cells gathering, seminiferous tubules disorder and dilated intercellular space. Furthermore, delayed spermatogenesis had occurred. The transcriptional changes of 19 genes along the hypothalamus–pituitary–gonadal–liver (HPGL) axis were investigated across the three organs. The disrupted expression of genes such as Ers, Ars, Vtgs, cyp19a, star, hsd3b, hsd17b3 and shh indicated the possible abnormal steroidogenesis, estrogenic or antiandrogen effects of PrP. Overall, the present results provided evidences for the toxigenicity and endocrine disruptive effects on the male mosquitofish of chronic PrP exposure, which highlights the need for more investigations of its potential health risks.