Snake venoms contain a large variety of proteins and peptides that affect the hemostasis and thrombosis. Numerous antithrombotic peptides have been found in snake venom. However, few studies have been performed on the proteolysis of snake venom for obtaining bioactive peptides. In this study, the Agkistrodon acutus venom was hydrolyzed using four commercial proteases (pepsin, papain, neutrase, and alcalase) and the hydrolysate was tested for antiplatelet aggregation activity. The pepsin hydrolysate, exhibiting the highest activity, was further purified by gel filtration and reversed-phase high performance liquid chromatography. A novel antithrombotic peptide SP-14 was obtained, and its sequence was identified as SHIHGDYSSPSGAP using tandem electrospray mass spectrometry. SP-14 inhibited U46619-induced rabbit platelet aggregation in a dose-dependent manner. It reduced the mortality of mice in acute pulmonary thrombosis model and decreased thrombus weight in rat arteriovenous shunt model, while with lower bleeding risk than aspirin. Therefore, SP-14 may be beneficial for new antithrombotic drug design and development.