The pathogenesis underlying alcoholic liver disease (ALD), which is often a result of alcohol abuse, currently remains unclear. Previous studies have reported that enteric dysbiosis serves an important role in the pathogenesis of ALD. The present study aimed to investigate the effects of glutamine and probiotics on a rat model of alcoholic liver disease (ALD). Sixty male Sprague-Dawley rats were randomly divided into 6 groups including control (C), alcohol (M), alcohol + Golden Bifido (T), alcohol + glutamine (G), alcohol + Medilac-S® (N) and alcohol + Golden Bifido + glutamine (L). Histology, body weight (BW), triglycerides (TG), serum aspartate transaminase (AST), alanine aminotransferase (ALT), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), diamine oxidase (DAO), occludin, endotoxin and D-lactate levels were assessed whilst changes in the gut flora were evaluated and compared. Results determined that all probiotic and glutamine treatments elevated the abnormally decreased BW and occludin levels whilst the abnormal elevated serum AST, ALT, TG, IL-6, TNF-α, DAO, endotoxin and D-lactate levels were significantly reduced following chronic ethanol consumption. Histopathological observation of the liver demonstrated that probiotic and glutamine treatments attenuated liver damage induced by alcohol. Moreover, sequencing determined that there was a reduction in Firmicutes as well as an increase in Actinobacteria, Proteobacteria and Porphyromonadaceae abundance in the ALD group compared with the healthy controls. However, these changes were prevented by glutamine and probiotic therapy. In conclusion, the present results suggested that probiotics and glutamine ameliorated ALD by suppressing inflammation and regulating the gut microbiota. Therefore, probiotic and glutamine treatments can potentially serve as therapies for the prevention and treatment of ALD.