Calcium-dependent protein kinases (CPKs) are key regulators of plant responses to abiotic stress; however, their roles in grapevine under cold stress remain poorly understood. In this study, we demonstrate that cold-repressed VpCPK9 and its counterpart NADPH oxidase VpRBOHD negatively regulate cold tolerance in grapevine. VpCPK9 interacts with VpRBOHD and phosphorylates VpRBOHD at Ser33, Ser132, and Ser138. This phosphorylation enhances VpRBOHD stability, thereby promoting reactive oxygen specie