© 2018 Published on behalf of the European Society of Cardiology. All rights reserved. Aims The effectiveness of cell-based treatments for regenerative myocardial therapy is limited by low rates of cell engraftment. Y-27632 inhibits Rho-associated protein kinase (ROCK), which regulates the cytoskeletal changes associated with cell adhesion, and has been used to protect cultured cells during their passaging. Here, we investigated whether preconditioning of cardiomyocytes, derived from human-induced pluripotent stem cells (hiPSC-CM), with Y-27632 improves their survival and engraftment in a murine model of acute myocardial infarction (MI). Methods and results After MI induction, mice were subjected to intramyocardial injections of phosphate-buffered saline, hiPSC-CM cultured under standard conditions (hiPSC-CM -RI), or Y-27632-preconditioned hiPSC-CM (hiPSC-CM +RI). The resulting engraftment rate calculated 4 weeks after implantation was significantly higher and the abundance of apoptotic transplanted cells was significantly lower in hiPSC-CM +RI recipients than in hiPSC-CM -RI animals. In cultured hiPSC-CM, Y-27632-preconditioning reversibly reduced contractile activity and the expression of troponin genes, while increasing their attachment to an underlying mouse cardiomyocyte (HL1) monolayer. Y-27632 preconditioning also increased the expression of N-cadherin and integrin ß1, the two cell junction proteins. hiPSC-CM +RI were also larger in cell area with greater cytoskeletal alignment and a more rod-like shape than hiPSC-CM -RI, both after transplantation (in vivo) and in culture. The effects of Y-27632 preconditioning on contractile activity and morphology of hiPSC-CMs in culture, as well as on their engraftment rate and apoptotic death in MI mouse grafts, could be recapitulated by hiPSC-CM treatment with the L-type calcium-channel blocker verapamil. Conclusion Preconditioning with the ROCK inhibitor Y-27632 increased the engraftment of transplanted hiPSC-CM in a murine MI model, while reversibly impairing hiPSC-CM contractility and promoting adhesion.