We conducted a case-control study to investigate the association of mitochondrial DNA (mtDNA) haplogroups with acute mountain sickness (AMS) in Han Chinese from southwestern (SW) China. Pearson’s chi-square test or Fisher’s exact test revealed significant reduction of mtDNA haplogroups D and M9, while a significant increase of haplogroup M7 in AMS subjects compared with non-AMS subjects. The multivariate logistic regression analysis after adjustment for body mass index (BMI), a risk factor of AMS in the present study, showed that both D and M9 were associated with significantly decreased risk of AMS, while M7 was associated with a significantly increased risk of AMS (OR = 0.605, p = 0.000; OR = 0.037, p = 0.001, and OR = 2.419, p = 0.001, respectively). In addition, the further analysis stratified by the AMS severities indicated that haplogroup B was correlated with a 2.41-folds increased risk of developing severe AMS (95%C.I = 1.288–4.514, p = 0.006). Our findings provide the evidence that, in SW Han Chinese, mtDNA haplogroups D and M9 are related to individual tolerance to AMS, while haplogroups M7 and B are risk factors for AMS.