The protein components of the venom obtained from Deinagkistrodon acutus in Yongzhou area of Hunan were identified and characterized using Nano-LC-ESI-MS/MS. The in vitro hemolysis of D. acutus venom was conducted with use of the conventional test tube method, and the LD50 of the venom was measured using a challenge test in a KM mouse model. Fifty types of proteins were identified, and the protein relative molecular mass are mainly distributed in 1.0×104~ 2.0×104(46.9%), 4.0×104~5.0×104(22.4%), 2.0×104~3.0×104(10.6%) and 7.0×104~8.0×104(7.6%) containing high-abundance of representative proteins including snake venom metalloproteinase acutolysin-A(11.7%), snake venom metalloproteinase H1(9.8%), thrombin-like enzyme acutobin-2(7.3%), antithrombin A subunit A(6.8%) and low-abundance proteins(＜0.1%) containing ecto-5'-nucleotidase, basic phospholipase A2 DAV-N6, growth/ differentiation factor 11. The minimal erythrocyte hemolysis concentration and LD50 of D. acutus venom were 60.00 μg/mL and 7.179 6 mg/kg, respectively. The typical clinical symptoms of challenged mice showed to be tachypnea, restlessness, unusual pruritus and widespread canker near the injection site and even to be repeatedly scratched, and death.