Abstract: This study delves into the significance of the long-chain acyl-CoA synthetase (ACSL) gene family in Gymnocypris przewalskii under saline-alkaline stress, conducting bioinformatics analysis on the genome date and exploring saline-alkaline responses through gene expression detection. The results showed that nine ACSL gene family members encoding proteins ranging from 640 to 834 amino acids. Despite notable differences in gene structure, motif and domain analysis showcased a high level of conservation among these members. Chromosome mapping revealed the dispersion of ACSL gene family members across nine chromosomes (Chr8, 13, 15, 40, 41, 48, 64, 82, and 87), all encoding hydrophilic properties. Except for ACSL2 and ACSL3a, the rest are stable proteins. The product of ACSL4b is an acidic protein, while all others are basic. Subcellular localization prediction results indicated that ACSL2 is localized to the cytoplasm, ACSL3a, ACSL3b, ACSL4a, and ACSL4b to the peroxisome, and the rest to the cell membrane. qRT-PCR analysis revealed tissue-specific expression patterns of ACSL genes in Gymnocypris przewalskii, with ACSL3b, ACSL4b, and ACSL5 being highly expressed in the kidney, while ACSL3a, ACSL4a, and ACSL5 exhibited elevated expression in the intestine. Under saline-alkali stress, high level expression of ACSL2, ACSL3a, and ACSL6 was detected in the gill, and transcription of ACSL1a, ACSL2, ACSL3a, and ACSL5 were markedly up-regulated in the kidney. In the intestine, expressions of ACSL3b and ACSL6 were prominently up-regulated. These findings highlight the ACSL gene family in Gymnocypris przewalskii contributes to multiple physiological functions under saline-alkaline stress, including energy supply through lipid metabolism, maintenance of osmotic balance, and immune regulation.