Trifloxystrobin is a systemic broad-spectrum foliar strobilurin fungicides that enters the aquatic environment during agricultural application. It is highly toxic and poses a potential risk to aquatic organisms, whereas the effect on the development of early life stages of fish are unclear. In this study, hatchability, time to hatching, and larval mortality were measured. Additionally, the expression of biomarker genes, including those involved in sex hormone pathways (er, vtg, cyp17, and cyp19a), thyroid hormone pathways (trα and dio2), and aryl hydrocarbon receptor pathways (ahr and cyp1a), was determined after embryos of medaka (Oryzias latipes) were exposed to different levels of trifloxystrobin (0, 0.1, 1, 10, and 100 μg/L) for 28 days. The results showed that there were significant differences between controls and the 100 μg/L treatment group in both hatchability and time to hatching of fertilized eggs (p < 0.05). Larval mortality was significantly increased in the 0.1, 1, and 10 μg/L treatment groups (p < 0.05). These results indicate that embryonic and larval development may be affected by trifloxystrobin exposure. Moreover, the mRNA levels of the er gene were significantly up-regulated at levels of trifloxystrobin above 1 μg/L treatment groups. Up-regulation of vtg, cyp17, and cyp19a mRNA levels was observed in the larvae at the lower concentration treatment groups. The mRNA levels of cyp1a genes were significantly up-regulated at all of the treatment groups. These results suggest that trifloxystrobin is a potential endocrine disruptor through effects on the sex hormone pathway and xenobiotic metabolism. The changes in cyp1a expression can be used as a highly sensitive biomarker to assess trifloxystrobin contamination in the early life stages of fish. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 648–655, 2015.