Japanese flounder (Paralichthys olivaceus) larvae were reared on feed supplement with Lactobacillus rhamnosus P15 alone and flavomycin. After 60 days of feeding, the total numbers of aerobes, vibrios, and lactic acid bacteria (LAB) were measured by plate colony count. The result showed that the numbers of LAB rose in both culture water and intestine of the flounder administrated with suspended and lyophilized bacteria. After 30 days, LAB colonized in the flounder gut with stable number. Meanwhile, the number of vibrios reduced, especially in the flounder gut, owing to the inhibitory effect of LAB. LAB had no effects on aerobes in culture water and Japanese flounder gut. The influences of LAB on the microflora in culture water and Japanese flounder gut were similar to those of flavomycin. It was suggested that LAB additive could be used in Japanese flounder culture as substitute for antibiotics.