The adult mammalian heart regeneration is largely prevented by the limited proliferative capacity of the resident cardiomyocytes (CMs). Here, the inventors identify Ephrin-B1 as a new critical regulator of adult CM proliferation. CM-specific transgenic repression of Ephrin-B1 promotes adult CM cell cycle reentry until division both in vitro and in vivo upon stimulation only, thus leading to substantial cardiac tissue regeneration through atypical CM proliferation and contractile function improvement to compensate for ageing stress, and apex resection. Cardiac deletion of efnb1 after myocardial infarction also improves considerably cardiac function and survival in mice. Together, these findings highlight Ephrin-B1 as a promising original target for future therapeutic strategies in cardiac regenerative medicine. Accordingly, the present invention relates to methods and pharmaceutical composition for cardiac regeneration based on use of Ephrin-B1 inhibitors.