Abnormal calcium handling is known to be instrumental in the development of cardiac hypertrophy and failure. Both isoforms 1 and 4 of the diastolic calcium extrusion pump plasma membrane calcium ATPase (PMCA) are downregulated in failing human and rodent hearts, yet their role in the disease process has not been studied. Methods: We generated cardiomyocyte-specific knockout mice for PMCA1 (PMCA1-/-), PMCA4 (PMCA4-/-) and the two pumps combined (PMCA1:4-/-), closely modelling the situation in human heart failure. Cardiac phenotype and calcium handling in isolated ventricular myocytes were studied in each mouse line under basal conditions, and in response to pressure overload following two weeks transverse aortic constriction (TAC). Results: Cardiac structure and function was not altered by the absence of PMCA1 or 4, nor their dual deletion under basal conditions. The PMCA contribution to global calcium extrusion was found to be 2%, and for the first time solely dependent upon PMCA1. When challenged with TAC, PMCA1-/- hearts rapidly progressed into decompensated hypertrophy displaying signs of impaired inotropy, lusitropy and heart failure as evidenced by decreased fractional shortening (20.6±1.6 v 32.4±3.3 %), increased relaxation time constant (8.8±0.8 v 6.6±0.3 ms) and greater normalised lung weight (11.5±1.1 v 7.8±0.8 mg/g) compared to controls. This was accompanied by increased apoptosis and extensive interstitial fibrosis. Analysis of intracellular calcium handling prior to the onset of failure (1 week post surgery) revealed impaired extrusion of the systolic transient in PMCA1-/- TAC cells compared to both wild type TAC and sham PMCA1-/- controls. In stark contrast, the deletion of PMCA4 attenuated the hypertrophic response to pressure overload with an 86% reduction in normalised heart weight compared to wild type TAC controls, whilst systolic and diastolic function were preserved. We hypothesised the deletion of PMCA4 may rescue the failing phenotype in PMCA1-/- mice, so PMCA1:4-/- mice also underwent TAC. Hearts displayed reduced heart weight/tibia length compared to wild type controls (7.0±0.4 v 9.2±0.6 mg/mm), accompanied by preserved function and reduced fibrosis. Conclusions: These results provide novel evidence of isoform-specific roles for PMCA1 and 4 in cardiac muscle, both capable of influencing the pathological progression of cardiac hypertrophy. PMCA1 may be critical in adapting to the increased demands placed on the heart during pressure overload, whilst the inhibition of PMCA4 may negate the need to adapt altogether and could be a potential target for future anti-hypertrophic therapies.