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Thermostable strains isolated from intestine of catfish were screened for their antagonism activity against Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC) and red head disease. Four thermostable strains were selected, and their antagonistic activity against E. ictaluri was compared. Adhesion to zebrafish larvae was determined as CFU/larva after exposure for 30min. The isolate Yab4 was found to be highly adhesive and was identified as Bacillus altitudinis based on a phylogenetic tree constructed using 16S rDNA and gyrB sequences. After 28d of feed supplementation with B. altitudinis Yab4, the growth performance of the treatment group is better than that of the control group, with a statistically significant difference observed between the two groups. Moreover, the innate immune indexes including alkaline phosphatase, lysozyme, and total proteins in serum increased significantly, and the expression levels of immune genes including IL-1β, TNF-α, and NF-κB p65 elevated significantly as well. Most importantly, the survival rate of the yellow catfish fed with B. altitudinis Yab4 supplementation reached 60%, marking a 35% increase compared to the control group when challenged with E. ictaluri HSN-1 infected intraperitoneally. This demonstrates that the potential of B. altitudinis Yab4 as a novel Bacillus probiotic to defend against E. ictaluri in catfish aquaculture through feed supplementation. [ABSTRACT FROM AUTHOR]