Alternagin-C (ALT-C) is a disintegrin-like protein purified from the venom of the snake, Rhinocerophis alternatus. Recent studies showed that ALT-C is able to induce vascular endothelial growth factor (VEGF) expression, endothelial cell proliferation and migration, angiogenesis and to increase myoblast viability. This peptide, therefore, can play a crucial role in tissue regeneration mechanisms. The aim of this study was to evaluate the effects of a single dose of alternagin-C (0.5 mg kg−1, via intra-arterial) on in vitro cardiac function of the freshwater fish traíra, Hoplias malabaricus, after 7 days. ALT-C treatment increased the cardiac performance promoting: 1) significant increases in the contraction force and in the rates of contraction and relaxation with concomitant decreases in the values of time to the peak tension and time to half- and 90% relaxation; 2) improvement in the cardiac pumping capacity and maximal electrical stimulation frequency, shifting the optimum frequency curve upward and to the right; 3) increases in myocardial VEGF levels and expression of key Ca2+-cycling proteins such as SERCA (sarcoplasmic reticulum Ca2+-ATPase), PLB (phospholamban), and NCX (Na+/Ca2+ exchanger); 4) abolishment of the typical negative force–frequency relationship of fish myocardium. In conclusion, this study indicates that ALT-C improves cardiac function, by increasing Ca2+ handling efficiency leading to a positive inotropism and chronotropism. The results suggest that ALT-C may lead to better cardiac output regulation indicating its potential application in therapies for cardiac contractile dysfunction.