The sarcomeric protein α-actinin-3, which is encoded by the ACTN3 gene, interacts with multiple proteins involved in structure, muscle metabolism, and signaling. α-Actinin-3 deficiency caused by a knockout or mutation of the terminator sequence of the ACTN3 gene leads to a change in muscle phenotype from fast-twitch fibers to slow-twitch fibers, resulting in enhanced aerobic metabolic energy, decreased calcium sensitivity, and degraded sprint and power performance. In this study, the single-nucleotide polymorphisms (SNPs) in the equine ACTN3 gene in Yili horses (n = 38) were detected and sequenced. Comparison of the obtained equine ACTN3 sequences with those in GenBank identified 15 SNPs, with one located in the promoter, eight located in exons, and six located in introns. All eight exonic SNPs, except for the missense mutation g.9059T>G, were synonymous. The g.9059T>G mutation lead to an increase in the free energy of the thermodynamic ensemble of the equine ACTN3 mRNA from −1157.84 to −1157.35 kcal/mol and changed the secondary structure of α-actinin-3, which may affect its function. We hypothesized that g.9059T>G might affect athletic performance and may be a candidate SNP for racehorse breeding.