Maintenance of a circumneutral intracellular pH is important for any organism. Acidophilic microorganisms thrive at low pH while maintaining their intracellular pH around 6.5. However, the mechanisms contributing to acidophile pH homeostasis are not well characterized. The authors investigated the proteomic response and cytoplasmic membrane fatty acid profiles of Acidithiobacillus caldus toward three pH values: 1.1, 2.5, and 4.0. Major rearrangements were observed but lower pH elicited larger changes. Differentially expressed transcription factors suggested tight transcriptional control of pH induced genes. Enzymes involved in sulfur metabolism were up-regulated at pH 1.1 suggesting either that: (1) cells required more energy for maintenance or (2) increased metabolic activity was a specific acid stress response to export intracellular protons via 1° electron transport proton pumps. Furthermore, glutamate decarboxylase, an important enzyme in Escherichia coli acid resistance, was uniquely expressed at pH 1.1. Other proteins previously shown to be involved in neutrophilic acid response, such as spermidine synthase, PspA, and toluene tolerance protein, were differentially expressed in At. caldus but require further investigation to show a direct link to pH homeostasis. Their roles in acidophilic organisms are discussed. Active modulation of fatty acid profiles was detected and suggested a more rigid membrane at low pH.