Tebuconazole is a widely used fungicide that has been detected in water ecosystems, of which the concentrations may affect the endocrine function of aquatic organisms. At present study, tissue-specific bioaccumulation of tebuconazole was found in ovary of adult zebrafish, indicating a potential risk of endocrine disruption. In order to evaluate the potential endocrine disrupting effects, three life stages (2 hpf (hours post-fertilization) −60 dpf (days post-fertilization), Stage I; 60–120 dpf, Stage II; 180–208 dpf, Stage III) of zebrafish (Danio rerio) were chronically exposed to tebuconazole at the concentrations ranging from 0.05 mg/L to 1.84 mg/L. Result showed that exposed to tebuconazole could lead to a male-biased sex differentiation in juvenile zebrafish and significant decrease of the percentage of germ cells in sexually-mature zebrafish. Egg production was significantly inhibited by 57.8% and 19.2% after Stage II- and Stage III-exposures, respectively. The contents of 17β-estradiol in gonad decreased by 63.5% when exposed to 0.20 mg/L tebuconazole at Stage II and by 49.5% after exposed to 0.18 mg/L tebuconazole at Stage III, respectively. For all stages exposure, reductions in 17β-estradiol/testosterone ratio were observed, indicating an imbalance in steroids synthesis. Additionally, tebuconazole reduced the expression of cyp19a, which was consistent with the decrease of E2 level. In overall, the present findings indicated that, playing as an anti-estrogen-like chemical, tebuconazole inhibited the expression of Cyp19, thereby impairing steroid hormones biosynthesis, leading to a diminished fecundity of zebrafish. Display omitted •Tebuconazole plays as an anti-estrogen endocrine disrupting effects on zebrafish.•Tebuconazole induces a male-biased gonadal differentiation in juvenile zebrafish.•Tebuconazole delays development of germ cells in puberty and adult zebrafish.•Block of E2 pathway may be the disrupting mechanism caused by tebuconazole. Tebuconazole blocks E2 pathway, thereby leading to a male-biased differentiation in juvenile zebrafish and delaying germ cells development in puberty and adult zebrafish, playing an antiestrogen-like role.