GPLD1 levels were elevated in HF patients and murine models. Cardiac-specific GPLD1 depletion exacerbated cardiac dysfunction and hypertrophy, while its overexpression ameliorated these effects, dependent on enzymatic activity. uPAR was identified as a potential binding target for GPLD1, and viral-mediated uPAR transduction completely abolished the protective effects of GPLD1 following TAC surgery. Mechanistically, GPLD1 was anchored to the plasma membrane and outer mitochondrial membrane via PI(4,5)P2 to cleave the GPI anchor of uPAR, thereby maintaining calcium homeostasis and mitochondrial function, and ultimately ameliorating cardiac dysfunction. Conversely, excess uPAR led to a decrease in PI(4,5)P2 levels, preventing the localization of GPLD1 to these membranes and causing it to disperse in the cytoplasm.