Objective To observe spontaneous liver tumor formation in Mdr2 knockout mice with C57BL/ 6 background. Methods Five wild type C57BL/ 6 mice and nine Mdr2 knockout C57BL/ 6-Abcb4^tm1 mice aged ( 11. 3 ± 4. 2)weeks were sacrificed after 65 weeks of continuous feeding. Serum and liver samples were collected and serum ALT, AST, and AFP levels were measured. Paraffin-embedded sections of liver tissue were stained with HE and Sirius red. CK-7 and CK-19 expression in tumor and adjacent tissues was detected by immunohistochemistry. Results Nine Mdr2 knockout mice formed liver tumors spontaneously. Serum ALT, AST, and AFP levels were significantly higher than those in wildtype mice(P< 0. 01). CK-7 and CK-19 expression was negative in Mdr2 knockout mice. Conclusions The Mdr2 knockout mice formed liver tumors spontaneously when continuously fed to ( 76. 3 ± 4. 2 ) weeks of age, and their pathological classification was hepatocellular carcinoma.