Abstract Under extreme environmental conditions such as hypoxia, insufficient nutrition, and glucose deficiency, the acetyl-CoA synthetase 2 (ACSS2)-mediated acetyl-CoA synthesis pathway plays an alternative role to ensure the normal operation of metabolic activities. To investigate the potential effect of the ACSS2 gene on hypoxic adaptation and its regulatory mechanism of gene expression in high-altitude cattle breeds, we analyzed the genetic variations of the ACSS2 gene in five Bos taurus taurus, Bos taurus indicus, hybrid Bos taurus taurus × Bos taurus indicus Chinese cattle breeds, and two Bos grunniens (yak) breeds distributed at different altitudes (95-3850 m). A total of 58 SNPs was detected in seven populations, and abundant genetic variation was found in high-altitude breeds. We identified the bovine ACSS2 core promoter region between g.-682 and g.-264 by using the luciferase assay in FFB and HepG2 cells. We also identified that the high-altitude hypoxia-specific haplotype (CAGTCT) was composed of six highly linked SNPs. The tagSNP g.-473 T>C (rs23) is located in the core promoter of ACSS2 in the Bos taurus taurus and yak breeds. The recombinant plasmid containing rs23 and analyses of luciferase activity of different genotypes showed that the activity of ACSS2 promoter increased significantly when T was mutated to C. We also found a yak-specific SNP rs20 that consists of 12 base insertions (g.-562 ins GAAAGGACCCTA) in the promoter of yak breeds. Luciferase activity analysis showed that the insertion mutant significantly decreased the promoter activity of ACSS2. Hence, ACSS2 may play an important role in the adaptation to high-altitude hypoxia by generating adaptive alleles to influence gene transcription in cattle. These results signify that different genetic variants and haplotypes affect the activity of the core promoter to regulate ACSS2 gene expression and subsequently overcome and adapt to a high-altitude environment within different cattle breeds. Our findings may have important implications for understanding the mechanism of adaptation to high altitude and for application of molecular breeding in Bos species.