In this study, we investigated the association between ACTN3 R577X polymorphism and non-acute ankle sprain by measuring the allele frequency and genotype distribution of ACTN3 in a Chinese Han population. We recruited 100 patients with non-acute ankle sprain and 100 healthy controls with no history of ankle injuries. Mass spectrometric analysis of single nucleotide polymorphism was used to analyze the genotype and allele frequencies of ACTN3. Results showed that the genotype frequency of RR in patients was 12.0%, which was significantly lower than that of the controls (24.0%) (OR = 1.7; 95%CI = 1.5-2.7; P = 0.001). The frequency distribution of the R allele in patients and controls were 68.5 and 56.7%, respectively (P = 0.002). Moreover, frequency of the RR genotype exhibited a downward linear trend with increased incidences of ankle sprain. Our results suggest that ACTN3 R577X polymorphism is associated with non-acute ankle sprain in the Chinese Han population.