(Bli) has been widely used as a probiotic and has positive effects on host health of different fish species. The ability to colonize the gastrointestinal tract is an important criterion for probiotic screening, but little attention has been paid to this character. To investigate the colonization characteristics of Bli-A1, full water exchange and feed sterilization with Co⁶⁰ irradiation were employed to avoid their influence on the gastrointestinal microbiome of . A high temperature selective culture method for Bli was assessed for its sensitivity and specificity, and then it was employed to investigate the dynamic characters of exogenous Bli-A1 in the intestine for 42 days with or without fasting. The spores of were used as an inert control to investigate the adhesion ability of Bli-A1 to mucosa in a feeding trial with Bli/Gs=186.4% in feed, and non-adhesion bacteria were removed by washing the intestinal wall. Then, Bil-A1/Gs on the intestinal content and wall were calculated. At last, the potential of Bli-A1 multiplying in the intestine was assessed with a sterile intestinal content culturing assay . With a full water exchange and feed sterilization, the concentration of Bli was reduced to <1 cfu/mL in water and not detected in feed (1.4×10⁴ cfu/g before irradiation). Selective culture for Bli-A1 was successful at 52℃ with a growth rate at 91.5±13.7% in water, 65.9±26.7% in 20% intestinal content homogenate, and 72.5±19.7% in 20% intestinal wall homogenate. It took 21 days for the inert Gs control to be completely excreted from the intestine after withdrawing exogenous supplement (=3, <1 cfu/intestine). When exogenous Bli-A1 supplement was withdrawn, the concentration of Bli-A1 in the intestinal content was sustained at~3.3×10² cfu/g for at least 42 days with continuous sterile feed supplement, whereas no Bli-A1 was detected at 14 days under fasting conditions. In the Bli-A1/Gs feeding trial, Bli-A1 in intestinal content was detectable to at least 28 days after exogenous Bli-A1 withdrawal. Adhesion-related Bli-A1 on the intestinal wall was undetectable at 7 days. At day 0, Bli-A1/Gs was equal to 197.9% in intestinal content, which was similar with the 186.4% in feed. Bli-A1/Gs was equal to 23.7% on the intestinal wall. Although Bli-A1 failed to adhere to mucosa and its adhesion ability was even weaker than that of the inert control, it grew well in intestinal content and reached a concentration of 2.0×10⁷ cfu/g Ictalurus punctatus long-term, unless the fish are fasted. That is because Bli-A1 possessed the ability to grow in intestinal content but fails to effectively adhere to mucosa.