Urotensin II (UTII) and its seven trans-membrane receptor (UTR) are up-regulated in the heart under pathological conditions. Previous in vitro studies have shown that UTII trans-activates the epidermal growth factor receptor (EGFR), however, the role of such novel signalling pathway stimulated by UTII is currently unknown. In this study, we hypothesized that EGFR trans-activation by UTII might exert a protective effect in the overloaded heart. To test this hypothesis, we induced cardiac hypertrophy by transverse aortic constriction (TAC) in wild-type mice, and tested the effects of the UTII antagonist Urantide (UR) on cardiac function, structure, and EGFR trans-activation. After 7 days of pressure overload, UR treatment induced a rapid and significant impairment of cardiac function compared to vehicle. In UR-treated TAC mice, cardiac dysfunction was associated with reduced phosphorylation levels of the EGFR and extracellular-regulated kinase (ERK), increased apoptotic cell death and fibrosis. In vitro UTR stimulation induced membrane translocation of β-arrestin 1/2, EGFR phosphorylation/internalization, and ERK activation in HEK293 cells. Furthermore, UTII administration lowered apoptotic cell death induced by serum deprivation, as shown by reduced TUNEL/Annexin V staining and caspase 3 activation. Interestingly, UTII-mediated EGFR trans-activation could be prevented by UR treatment or knockdown of β-arrestin 1/2. Our data show, for the first time in vivo, a new UTR signalling pathway which is mediated by EGFR trans-activation, dependent by β-arrestin 1/2, promoting cell survival and cardioprotection.