Abstract: Bacteria 4-L-16 was isolated from natural cake fertilizer fermentation liquid, in order to evaluate its anti- fungi activity and colonizing regulation systematically. Nine kinds of pathogen including Fusarium oxyspum f. sp.cubense Race 4 were taken as indicative strains to detect the anti-fungi activity of antagonistic bacteria by the methods of plate confrontation and containing medium. Bacteria 4-L-16 was also identified by Gram staining, morphologic observation, physiological and biochemical characteristics analysis and 16S rDNA sequence analysis. The results showed that the strain 4-L-16 had broad-spectrum resistance to the nine kinds of pathogen, the inhibition rates of mycelium growth were 28.05%-76.07%, and the inhibition rates of spore germination were 24.44%-70.00%. For banana Fusarium wilt, the inhibition rates of mycelium growth and spore germination were 76.07% and 70% , respectively, and this strain was identified as Bacillus methylotrophicus. By the method of antibiotic marker, anti 600 μg/mL rifampicin marked strain of 4-L-16 was obtained. The pot test showed that the strain 4-L-16 could colonize in banana rhizosphere soil and banana plant, after inoculating 10 d, the soil colonization reached a maximum quantity of 6.67×106 CFU/g. But with the extension of days, the number of colonization decreased gradually, the quantity tended to be stable of 2.05×106 CFU/g after inoculating 25 d. The colonization mostly took place in the root of banana, was 4.38×103 CFU/g. Bacillus methylotrophicus 4-L-16 was taken as a new microbial resource for biological control of Banana Fusarium wilt disease for its broad-spectrum resistance activities and good colonization abilities.