Protozoan parasites of the phylum Apicomplexa actively move through tissue in order to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase protein (TgGC), which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca(2+)] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca(2+) ionophore. Further, we found that TgGC is required for transducing changes in extracellular pH and [K(+)] to activate cytosolic [Ca(2+)] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca(2+) signaling and motility in Toxoplasma.