This study was aimed to investigate the impact of probiotics on regulating the ROS/JNK signaling pathway their underlying mechanism of action in the treatment of nonalcoholic fatty liver disease. For this purpose, male C57BL/6 mice were randomly divided into three groups: control, probiotics, and model groups. Methionine and choline deficiency (MCD) diets were fed for four weeks to establish a NAFLD mouse model. Serum levels of ALT, AST, TC, and TG were detected. Moreover, the pathological changes of the liver and ileum tissues were observed by hematoxylin and eosin (H&E) staining, and the content of reactive oxygen species (ROS) in liver tissues was determined. In addition, the levels of D-lactic acid and plasma and small intestine diamine oxidase were measured to evaluate the effects of probiotics on the intestinal tract of NAFLD mice. The expression levels of p-JNK, Bax, and Caspase-3 were established to analyze the regulatory mechanism of probiotics on the JNK signaling pathway. We found that probiotics improve liver function, repair intestinal barrier and significantly suppressed oxidative stress, JNK phosphorylation. Moreover, the application of probiotics regulated the expression of signaling pathway-related proteins and promoted the intestinal barrier function repair and decreased intestinal permeability. The data above suggest that probiotics alleviate NAFLD, whose mechanism might be associated with the regulation of ROS/JNK signaling pathway and the suppression of oxidative stress and apoptosis.