Acetochlor (ACT) is a chiral chloroacetamide pesticide that has been heavily used around the world, resulting in its residues being frequently found in surface waters. It has been reported that ACT is an endocrine disrupting chemical (EDC) with strong thyroid hormone-disrupting activity in aquatic organisms. However, the enantioselectivity underlying thyroid disruption has yet to be understood. In this study, using a zebrafish embryo-larvae model, the enantioselective thyroid disruption of ACT was investigated at a series of environmentally relevant concentrations (1, 2, 10 and 50 μg/L). Our results showed that both racemic ACT and its enantiomers significantly increased the malformation rates of embryos at 72 h postfertilization (hpf). Decreased thyroxine (T4) contents and increased triiodothyronine (T3) contents were found in larvae at 120 hpf, with (+)-S-ACT exhibiting a greater effect than (-)-R-enantiomer. Similarly, (+)-S-ACT also showed a stronger effect on the mRNA expressions of thyroid hormone receptors (TRα and TRβ), deiodinase2 (Dio2) and thyroid-stimulating hormone-β (TSHβ) genes. The observed enantioselectivity in TR expressions was consistent with that of in silico binding analysis, which suggested that (+)-S-enantiomer binds more potently to the TRs than (-)-R-enantiomer. In general, ACT enantiomers showed different influences on the secretion of THs, expression of TH-related key genes and binding affinity to TRs. Considering the different toxicity of different enantiomers, our study highlights the importance of enantioselectivity in understanding of thyroid disruption effects of chiral pesticides.