Abstract: Background　Understanding molecular mechanisms of high-altitude adaptation is crucial for combating mountain sickness. While native highlanders exhibit distinct genetic/epigenetic traits, systematic whole-genome methylation comparisons across acclimatization stages are lacking. Objective　To investigate the differences in DNA methylation among populations in plain and plateau environments and analyze their functional implications. Methods　A self-established high-altitude research cohort was composed of native lowlanders (NLs), acclimatized newcomers (ANs), and native highlanders (NHs). Whole-genome bisulfite sequencing (WGBS) was used to obtain their methylation data. Differential methylated regions (DMRs) analysis and epigenomewide association studies (EWAS) were conducted among the populations to identify differential methylation signals between the groups. GO and KEGG enrichment analyses were then performed to elucidate the biological functions of these methylation differences. Results　This study included 117 NLs (all men, aged 20.15 ± 1.43 years), 162 ANs (all men, aged 22.07 ± 2.65 years), and 124 NHs (101 men and 23 women, aged 22.10 ± 7.34 years). After entering the plateau, the methylation levels of NLs decreased genome-wide (P＜0.01). Compared with NLs, ANs exhibited 440 DMRs and 611 differentially methylated CpGs (DMCs), driving significant changes in immune functions (such as T cell activation and NK cell cytotoxicity pathways) and energy metabolism patterns (e.g., the LDHC gene), but also accompanied by abnormal activation of pathways related to cardiomyopathy. Compared with ANs, NHs had 1 313 DMRs and 2 022 DMCs, with differences mainly enriched in the calcium signaling pathway and gene regions such as EPAS1/EGLN1. The overall methylation level of NHs was significantly lower than that of NLs, and NHs had 247
DMRs and 688 DMCs, with differences mainly enriched in the HIF-1 pathway (q＜0.01) and the autophagy signaling pathway. Conclusion　DNA methylation plays an important role in human adaptation to high-altitude environments. In comparison, NHs not only exhibit a significantly lower overall methylation level than NLs, but also display a greater number of specific differentially methylated regions, suggesting that they may achieve long-term stable adaptation to hypoxic environments and cellular homeostasis through precise regulation of the methylation status of key genes.