Probiotics are widely used in aquaculture. This article aims to study the effect of Bacillus amyloliquefaciens LSG2‐8 on the intestinal barrier function of Rhynchocypris lagowskii. B. amyloliquefaciens LSG2‐8 were added to R. lagowskii basal diets (CK) as additives at four concentrations: 1.0 × 106 (D‐6), 1.0 × 107 (D‐7), 1.0 × 108 (D‐8) and 1.0 × 109 (D‐9) CFU g−1 by dry weight of basal diet. After a 56‐day feeding experiment, the activities of intestinal digestive enzymes and immunity‐related enzymes of R. lagowskii on group D‐6, D‐7, D‐8 and D‐9 diet were significantly higher than the control (P < 0.05). In molecular experiments, the authors found that the levels of TGF‐β mRNA, IL‐10 mRNA, ZO‐1 mRNA and claudin‐3 mRNA in group D‐8 R. lagowskii were significantly higher (P < 0.05) than those of the control and other groups. Furthermore, the levels of IL‐1β and IL‐8 mRNA of R. lagowskii on group D‐6, D‐7, D‐8 and D‐9 diet were significantly lower than those of the control (P < 0.05). In addition, the authors found that B. amyloliquefaciens LSG2‐8 can regulate the intestinal flora balance and improve the intestinal structure of R. lagowskii. In conclusion, B. amyloliquefaciens LSG2‐8 can improve the intestinal barrier function of R. lagowskii and can be used as a feed additive in aquaculture. [ABSTRACT FROM AUTHOR]