Calcium (Ca )-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca sensor/kinase-effector proteins with diverse functions in plants. In , CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intermolecular autophosphorylation on Ser318 and can additionally be transphosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca activation at physiological concentrations of Ca , possibly through stabilization of the Ca -bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular Ca to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem elongation, indicating pathway-specific requirements for phosphorylation-based Ca -sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs, which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.