Genetic factors are closely related to the high-altitude adaptation. Several candidate genes have been reported to be significantly associated with high-altitude adaptation. Megakaryocytic leukemia 1 (MKL1) is a transcriptional regulator, which plays crucial roles in the phenotypic modulation of smooth muscle cells. To explore whether MKL1 gene is involved in high-altitude adaptation, we performed genetic association study between MKL1 gene polymorphisms and high-altitude adaptation. We recruited 595 Tibetans from the Qinghai-Tibet plateau and 442 Hans living at low altitude. The SNP rs59098711 locus in the MKL1 gene was genotyped using the MassARRAY assays. We compared the genotypes and allele frequency distributions of rs59098711 between the Hans and Tibetans. The results showed that the genotypes of rs59098711 are significantly different between these two populations (P<0.05). We further compared the genotypes of rs59098711 between Tibetans and other ethnic groups using data from various public databases. We also observed that the genotypes of rs59098711 are significantly different between Tibetans and these other populations (P<0.05). Bioinformatics analyses suggested that rs59098711 could be located at the enhancer region and regulate the expression of the MKL1 gene. Additional study of gene expression data from available public datasets showed that MKL1 gene was up-regulated in a hypobaric hypoxic environment. Our results suggested that the genetic polymorphism rs59098711 locus of the MKL1 gene is associated with high-altitude adaption.