Studies of acid mine drainage (AMD), a typically extremely acidic environment, can provide opportunities to determine how microorganisms adapt to low pH environments by regulating membrane lipid compositions. In this study, to gain an understanding of microbial pH adaptation mechanisms, we investigated the distribution of lipid biomarkers, primarily of microbial origin, in an AMD site at Dexing copper mine, Jiangxi, China. Microbial lipid biomarkers, including glycerol dialkyl glycerol tetraethers (GDGTs), monoalkyl glycerol ethers, fatty acids (FAs), 3-hydroxy fatty acids (3-OH-FAs), sterols, and hopanoids, were detected in surface and 10-cm-deep core sediments. Branched FAs and 3-OH-FAs were found to occur at low levels in the extremely low pH conditions. Similarly, there were fewer methyl moieties in branched GDGTs detected in the AMD, as reflected by the lower methylation degree of branched tetraethers, indicating that a reduction in membrane lipid methyl moieties may be a common strategy whereby bacteria adapt to low pH environments. The presence of only small amounts of crenarchaeol in the AMD suggests that the extremely acidic conditions are inimical to the growth of Thaumarchaeota. In contrast, abundant Euryarchaeota inhabit the AMD and produce an isoprenoid GDGT (isoGDGT) profile dominated by GDGT-2, along with GDGT-5 and GDGT-6, which are rarely detected in non-extreme environments. Furthermore, the GDGT-2/(GDGT-1 + GDGT-3) ratio in the AMD was found to be significantly higher than that in soils and was negatively correlated with pH. These findings indicate that increases in the cyclopentyl rings of isoGDGTs may be favorable for maintaining an osmotic balance across archaeal cell membranes. All the modifications adopted by bacteria and archaea result in more condensed cell membranes.