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Backgrounds and aims Aluminum contamination of water is becoming increasingly serious and threatens the health status of fish. Lactobacillus plantarum CCFM639 was previously shown to be a potential probiotic for alleviation aluminum toxicity in Nile tilapia. Considering the significant role of the gut microbiota on fish health, it seems appropriate to explore the relationships among aluminum exposure, probiotic supplementation, and the gut microbiota in Nile tilapia and to determine whether regulation of the gut microbiota is related to alleviation of aluminum toxicity by a probiotic in Nile tilapia. Methods and results The tilapia were assigned into four groups, control, CCFM639 only, aluminum only, and aluminum + CCFM639 groups for an experimental period of 4 weeks. The tilapia in the aluminum only group were grown in water with an aluminum ion concentration of 2.73 mg/L. The final concentration of CCFM639 in the diet was 108 CFU/g. The results show that environmental aluminum exposure reduced the numbers of L. plantarum in tilapia feces and altered the gut microbiota. As the predominant bacterial phyla in the gut, the abundances of Bacteroidetes and Proteobacteria in aluminum-exposed fish were significantly elevated and lowered, respectively. At the genus level, fish exposed to aluminum had a significantly lower abundance of Deefgea, Plesiomonas, and Pseudomonas and a greater abundance of Flavobacterium, Enterovibrio, Porphyromonadaceae uncultured, and Comamonadaceae. When tilapia were exposed to aluminum, the administration of a probiotic promoted aluminum excretion through the feces and led to a decrease in the abundance of Comamonadaceae, Enterovibrio and Porphyromonadaceae. Notably, supplementation with a probiotic only greatly decreased the abundance of Aeromonas and Pseudomonas. Conclusion Aluminum exposure altered the diversity of the gut microbiota in Nile tilapia, and probiotic supplementation allowed the recovery of some of the diversity. Therefore, regulation of gut microbiota with a probiotic is a possible mechanism for the alleviation of aluminum toxicity in Nile tilapia.