The α-actinin-3 (ACTN3) gene rs1815739 (C/T, R577X) polymorphism is a variant frequently associated with athletic performance among different populations. However, there is limited research on the impact of this variant on athlete status and physical performance in basketball players. Therefore, the aim of this study was twofold: (1) to determine the association ofACTN3rs1815739 polymorphism with changes in physical performance in response to six weeks of training in elite basketball players using 30 m sprint and Yo-Yo Intermittent Recovery Test Level 2 (IR 2) tests, and (2) to compareACTN3genotype and allelic frequencies between elite basketball players and controls. The study included a total of 363 individuals, comprising 101 elite basketball players and 262 sedentary individuals. Genomic DNA was isolated from oral epithelial cells or leukocytes, and genotyping was performed by real-time PCR using KASP genotyping method or by microarray analysis. We found that the frequency of theACTN3rs1815739 XX genotype was significantly lower in basketball players compared to controls (10.9 vs. 21.4%,p= 0.023), suggesting that RR/RX genotypes were more favorable for playing basketball. Statistically significant (p= 0.045) changes were observed in Yo-Yo IRT 2 performance measurement tests in basketball players with the RR genotype only. In conclusion, our findings suggest that the carriage of theACTN3rs1815739 R allele may confer an advantage in basketball.