Abstract NADK is the sole cytosolic enzyme responsible for synthesizing NADP+ from NAD+ within cells. The homeostasis of NAD+/NADP+ is controlled by NADK, usually dysregulated in various cancers, yet the precise underlying regulatory mechanisms remain largely unknown. In this study, we discover that PRMT6 methylates NADK at R39, R41, and R45, resulting in the suppression of NADK kinase activity and NADP+ synthesis. Mutations of these sites promote cancer cell proliferation and tumor growth.