Acidophiles are the organisms that usually grow at a pH of 3.0 or below. They usually occur in an environment rich in iron and sulfur. These organisms have the ability to oxidize sulfur and iron producing sulfuric acid thus making the environment acidic. The environments where acidophiles are commonly found are termed acid mine drainage (AMD) or acid rock drainage (ARD). The production of acid helps in the dissolution of several minerals present in the environment; hence acidophiles play important roles in bio-metallurgy. Acidophiles are a diverse group of organisms belonging to all three domains of life viz. Bacteria, Archaea to Eukarya. Many of them are obligate chemolithotrophs, and few are acidophilic heterotrophs. Usually, the chemolithotrophs are the ones that oxidize ferrous iron and sulfur into ferric iron and sulphate respectively. During their growth, they produce or secrete organic waste products, which are otherwise toxic to obligate chemolithotrophs but are usually scavenged by the acidophilic heterotrophs. Because of the acidic environment, proton concentration [H+] is always high outside the cell compared to the cytoplasm, thus pH gradient across the membrane is readily generated for these organisms. The pH gradient so generated forms proton motive force (PMF), which is utilized for the coupling of ADP and Pi to generate ATP molecules with the help of ATPase enzymes. However, continuous flow of proton from outside into the cell results in the cytoplasmic protonation or acidification of cytoplasm which may lead to deleterious effects such as denaturation or inactivation of several macromolecules such as DNA or proteins. Thus, the acidophiles must have evolved mechanism(s) to resist or tolerate low pH. Several mechanisms, such as proton impermeability, reverse membrane potential, etc. have been proposed to explain their ability to thrive under low pH maintaining the homeostatic balance in their systems. In this chapter, the diversity of acidophilic microorganisms and the mechanisms of their acid resistance are discussed in detail.