As tricresyl phosphate (TCrP) is commonly found in global water sources, its potential reproductive toxicity to fish is of increasing concern. Japanese medaka larvae were exposed to TCrP at 657.9, 1,511, and 4042 ng/L for 100 days. We identified significant fertilization inhibition (6.9%-12.8%) in all exposure groups. Intersex was significantly induced at 4042 ng/L, with an incidence of 22.0%. TCrP exposure also caused dilation of the efferent duct in the testes with maximum duct widths of 83.3, 93.2, and 149.7 μm in the 657.9, 1,511, and 4042 ng/L exposure groups, respectively. These widths were all significantly larger than that observed in the control group (37.7 μm) and likely contributed substantially to fertilization inhibition. The TCrP metabolites 4-OH-MDTP and 3-OH-MDTP, were detected at high concentrations in the liver and elicited 5.8-fold and 5.3-fold greater androgen receptor antagonistic activity than that elicited by TCrP (39.8 μM), which may explain the intersex observed in low exposure groups. 4-OH-MDTP and 3-OH-MDTP elicited anti-estrogenic activities by blocking the estrogen receptor, and the concentrations at which its responses were equal to the IC20 of tamoxifen were 16.1 μM and 18.9 μM, respectively, as detected using the yeast two-hybrid assay. Such anti-estrogenic activities were likely the main driver of dilation of the efferent duct. Observed adverse outcomes after exposure to TCrP all occurred under environmentally relevant concentrations, suggesting considerable ecological risk to wild fish.