A large proportion of intracellular Ca2+ in Toxoplasma gondii tachyzoites is stored within acidocalcisomes. These organelles are characterized by their acidic nature and high calcium and polyphosphate (polyP) content. The activity of a Ca2+/H+-ATPase named TgA1 may be important for the accumulation of Ca2+ in these organelles. This enzyme belongs to a group of plasma membrane Ca2+-ATPase (PMCA) that lack a calmodulin-binding domain and have vacuolar localization. To investigate the role of this enzyme, we have generated T. gondii mutants deficient in TgA1 through gene disruption. Proliferation of these mutants decreased dramatically because of deficient cell invasion. In addition, these cells had reduced virulence in a mouse model. Biochemical analysis revealed that the cell polyP content was drastically reduced, and the basal calcium levels were increased and unstable. Microneme secretion under the conditions of stimulation by ionophores was altered. Complementation of null mutants with TgA1 restored most functions. In summary, these results establish a link between TgA1, calcium homeostasis, polyP storage and virulence.