Cardiac hypertrophy and heart failure are affecting almost one million people in the UK alone. The available therapies of cardiac hypertrophy are for symptomatic treatment. Recently attention has been moved towards identification of novel drugs which intervene with signalling pathways involved in hypertrophy. To achieve this goal it was important to understand the role of genes involved in the development of cardiac hypertrophy. One of such genes is plasma membrane calcium ATPase isoform 4 (PMCA4). Although several studies which used genetically modified animal models suggested the involvement of PMCA4 during the development of cardiac hypertrophy, the actual role of PMCA4 is still unclear. In this study, we will clarify the role of PMCA4 during the development of cardiac hypertrophy using a novel PMCA4 specific inhibitor. Until now there is no known PMCA4 specific inhibitor so a library of 1280 medically optimised compounds was screened using a novel in vitro assay which measures the ATPase activity of PMCA4. The compound aurintricarboxylic acid (ATA) was identified, which inhibited PMCA4 ATPase activity with higher affinity (IC50= 100 nM) compared with related ATPases. In isolated neonatal rat cardiomyocytes, ATA showed dose dependent inhibition of phenylephrine-induced hypertrophy. In vivo studies showed that ATA (5mg/kg body weight/day IP) significantly reduced the development of pressure-overload induced hypertrophy in wild type mice following two weeks transverse aortic constriction (TAC). Echocardiography and haemodynamic analyses showed that ATA treatment significantly reduced the abnormal left ventricular remodelling after TAC compared with vehicle treatment. ATA treated TAC mice showed a significant reduction in the enlargement of heart weight/tibia length ratio as well as cardiomyocyte cross sectional surface area compared with vehicle treated TAC mice. A significant reduction in the expression of the hypertrophic markers ANP and BNP and, importantly, in the percentage of fibrosis was observed in ATA treated TAC mice compared with vehicle treated TAC mice. In addition, ATA treatment significantly reversed the already established pressure overload induced hypertrophy following three weeks TAC. ATA treatment to TAC mice led to a significant reduction in the expression of the bona fide calcineurin target MCIP1 and a reduction in NFAT phosphorylation level in vivo and NFAT transcriptional activity in vitro. ATA did not show a direct inhibition to the active form of calcineurin nor to the phosphatase activity of full length calcineurin.In conclusion, we have identified ATA as a novel and specific inhibitor to PMCA4 ATPase activity. Pharmacological inhibition of PMCA4 significantly reduces the hypertrophic response to pressure overload likely through inhibition of calcineurin/NFAT signalling.