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Leishmania sp. belong to the trypanosomatid group of protozoan parasites and are responsible for a wide variety of diseases collectively known as leishmaniasis. They continue to be a major health problem of the world threatening ~ 350 million people, majority of whom are from the impoverished sections of the community. Unavailability of a vaccine and increasing signs of resistance to few existing drugs warrants consolidated effort towards discovery of novel antileishmanial drug targets. In recent times, carbonic anhydrases (CAs) have been studied in a number of pathogenic microorganisms and preliminary evidences suggest that it can be a promising drug target. We discovered that Leishmania major expresses two CAs (LmCAs), namely a β-family LmCA1 and an α-family LmCA2. Our preliminary pharmacological inhibition studies, using an established CA inhibitor, suggested that LmCA activity is important for parasite growth and survival. Coupling pharmacological inhibition data with structural insights obtained from homology model of LmCA1, we arrived at the conclusion that LmCAs, especially LmCA1, is structurally distinct from its human α-CA counterparts. These encouraging results prompted us to investigate the role of LmCA1 in parasite physiology. Subcellular localization studies confirmed that LmCA1 is localized in the cytosol of the parasite. LmCA1-/- knockout strain could not be generated in repeated attempts, suggesting its functional indispensability to the parasite. However, we were able to generate LmCA1+/- heterozygous strain, which grew normally in neutral medium but suffered from intracellular acidosis-induced cell cycle arrest, growth retardation and morphological abnormalities in acidic pH. This strain also showed reduced parasite load and infectivity in macrophage cell line and mice. Thus, LmCA1 is crucial for cytosolic buffering of L. major and plays a determining role in parasite proliferation in acidic milieu in vitro as well as within phagolysosomes of host macrophages. In addition to acid acclimatization, LmCA1 plays an indispensable role in the gluconeogenic pathway of the parasite. Under low glucose stress, LmCA1+/- heterozygous strain suffered from cell cycle arrest, growth retardation and morphological abnormalities. These phenotypes were due to depleted intracellular glucose pool, which ultimately resulted in faulty ATP production in the mutant parasite. Thus, indispensability of LmCA1 in such crucial physiological and metabolic processes of Leishmania highlights LmCA1 as a potential antileishmanial drug target.