Objective To investigate the mechanism of probiotics in protecting rats with acute intrahepat⁃ic cholestasis induced by alpha⁃naphthylisothiocyanate（ANIT）. Methods Forty Sprague⁃Dawley（SD）rats wererandomly divided into 4 groups：control group，model group，model + probiotics group and nomal + probioticsgroup. Probiotics 5 × 107 CFU/（kg·d）was administrated to the rats in the model + probiotics group and normal +probiotic group for two weeks and normal saline 5 mL/（kg·d）to the model group and control group. After adminis⁃tration and fasting for 4 h on the twelfth day，the model group and model + probiotics group were intragastricallyadministrated with ANIT（75 mg/kg）for modeling and the control group and normal + probiotics group were witholive oil. At 48 h after modeling，all rats were executed for specimen collections. The concentrations of the serumtotal bile acid（TBA），alanine aminotransferase（ALT），aspartate aminotransferase（AST），total bilirubin（TBiL）and direct bilirubin （DBiL） were determined. The levels of malondialdehyde （MDA），superoxide dismutase（SOD），tumor necrosis factor⁃α（TNF⁃α），interleukin⁃6（IL⁃6）and interleukin⁃1β（IL⁃1β）in the liver tissueswere detected. The expressions of Toll⁃like receptor 4（TLR4）and nuclear factor kappa B（NF⁃κB）in the livertissues were determined by real⁃time fluorescent quantitative RT⁃PCR and Western Blot（WB）. Results Comparedwith the normal control group，the levels of serum TBA，TBil，DBil，ALT and AST，the contents of MDA，TNF⁃α，IL⁃6 and IL⁃1β，and the expressions of mRNA and protein of TLR4 and NF⁃κB in the liver tissues in the modelgroup were significantly increased（P < 0.05），while the activity of SOD in the liver tissues was significantlydecreased（P < 0.05）. Compared with the model group，probiotics could reduce the levels of TBA，ALT，AST，TBiL，DBiL，TNF⁃α，IL⁃6 and IL⁃1β（P < 0.05），increase the activity of SOD（P < 0.05），meanwhile signifi⁃cantly inhibit the expressions of TLR4 and NF ⁃κB mRNA and protein in the liver tissues（P < 0.05）. Conclusion Probiotics protect intrahepatic cholestatic rats by regulating the signal pathway of TLR4/NF⁃κB，and further inhibit⁃ing inflammation and oxidative stress.