Objective To establish rat models of extrahepatic biliary atresia, and to observe the characteristics of 99Tcm-MIBI hepatobiliary scintigraphy and evaluate its association with the expression P-glycoprotein (P-gp) in intestinal tissues. Methods A total of 12 SD rats were randomly divided into the normal control group (3 rats) and the group of common bile duct ligation (CBDL; 9 rats). CBDL was used to establish the rat model of extrahepatic biliary atresia. 99Tcm-MIBI hepatobiliary scintigraphy was performed at 2, 3 and 4 weeks after ligation in the CBDL group and normal control group with continuous dynamic acquisition (3 min/frame) for 30 min and then delaying imaging at 30 min, 1, 2 and 3 h. After that, all rats were sacrificed, and the blood samples were taken out for the determination of serum ALT, AST, TBIL, DBIL, IBIL, ALP, γ-GT and TBA, and the tissues of duodenum, jejunum, ileum, colon and cecum were taken out for analyzing the expression level of P-gp by immunohistochemistry. Two-sample t test and one-way analysis of variance were used. Results Compared with the normal control group, the serum levels of ALT, AST, TBIL, DBIL, IBIL, ALP, γ-GT and TBA were significantly increasing at 2, 3, 4 weeks after ligation in CBDL group (t: -3.04 to -44.54, all P 0.05). Conclusion 99Tcm-MIBI can be excreted through intestinal mucosa, and this process may be associated with P-gp expression. Key words: Biliary atresia; P-glycoprotein; Radionuclide imaging; Rats; MIBI