Purinergic P2Y6 receptor (P2Y6R) is a member of G protein-coupled receptor (GPCR) activated by uridine nucleotides. We previously reported that MRS2578, a specific inhibitor of P2Y6R, suppressed cardiac remodeling and dysfunction induced by pressure overload (EMBO J, 2008). We thus hypothesized that P2Y6R deficient mice would show the same phenotype and performed transverse aortic constriction (TAC) to induce cardiac pressure overload in P2Y6R deficient mice. Contrary to our expectation, P2Y6R deficiency exacerbated cardiac remodeling induced by pressure overload. Surprisingly, transgenic mice with cardiomyocytes-specific overexpression of P2Y6R was also found to worsen cardiac remodeling after pressure overload. We thus speculated that MRS2578 could activate P2Y6R-dependent cardioprotective signaling pathway. We found that MRS2578 upregulated survival factors in mouse hearts, such as SOD2 and SIRT3 in a P2Y6R-dependent manner. We further found that MRS2578 treatment multimerized P2Y6R proteins, which were translocated from plasma membrane to cytosol. These results suggest that MRS2578-induced changes in localization and protein quality of P2Y6R is essential for the activation of cardioprotective signaling pathways.