Plasma membrane H⁺-ATPases play important roles in plant growth by keeping a proton gradient across the membrane. Although cold or pathogen stress affects the activity of the H⁺-ATPase and plant growth, its regulatory mechanisms are not fully elucidated. A rice 55-kDa calcium-dependent protein kinase was activated on cold treatment and pathogen infection. Here we showed that the kinase phosphorylated two Thr residues in the auto-regulatory domain of the H⁺-ATPase in vitro. Phosphorylation of these Thr residues might contribute to the ATPase activity regulation during cold or pathogen stress.