Abstract: To investigate the protective effects of fish-derived Lactobacillus acidophilus AC on juvenile zebrafish (Danio rerio) under ammonia nitrogen stress, a total of 180six-week-old AB strain juvenile Danio rerio mean weight (83.02±4.08) mg were randomly divided into four groups: AC0 group (0 CFU/mL Lactobacillus acidophilus AC), AC5 group (10⁵ CFU/mL), AC6 group (10⁶ CFU/mL), and AC7 group (10⁷ CFU/mL). The bacteria were added to the rearing water for 32d, after which fish were exposed to 35.80 mg/L total ammonia nitrogen for 48h. The gill antioxidant parameters, gill tissue pathological changes, and the expression of related genes in gill and brain tissues were examined. Antioxidant parameters analysis of the gill tissues revealed that the activity of Na⁺/K⁺-ATPase and total superoxide dismutase (SOD) was significantly higher in the AC7 group than that in the AC0 (P<0.05), while the malondialdehyde (MDA) content was significantly lower (P<0.05). Pathological analysis of the gill tissues showed varying degrees of damage across groups. The AC0 group exhibited severe gill damage, characterized by curling of the gill filaments and noticeable edema, degeneration, and necrosis of pillar epithelial cells at the base of lamellae. In contrast, the experimental groups exhibited milder gill damage. Gene expression analysis showed that inflammatory cytokine genes were generally downregulated in treated fish compared to the control group, with the brain factor-α (TNF-α) expression in AC7significantly lower than that in the AC0 (P<0.05). In gill tissue, expression of glutamate dehydrogenase (GDH) and SOD was significantly higher in AC7 than that in AC0 and AC5 (P<0.05), while glutamine synthetase (GS) expression in both gill and brain was significantly higher in AC7 than that in the AC0 and AC5 (P<0.05). Additionally, B-cell lymphoma-2 (Bcl-2) expression in gill tissues was significantly upregulated in AC7 relative to other groups (P<0.05), and caspase 8 expression in gill and brain tissues was downregulated to varying degrees in all treated groups. In conclusion, Lactobacillus acidophilus AC alleviates oxidative stress damage in the gills by regulating the expression of antioxidant and ammonia assimilating enzyme genes, thus helping juvenile Danio rerio resist ammonia nitrogen stress.