Casein glycomacropeptide (GMP) is known to promote the in vitro growth of Bifidobacteria and Lactobacilli. In this paper, we used conven- tional culture techniques and fluorescent in situ hybridization (FISH) techniques to investigate the effect of casein GMP on mice fecal microbiota. The population structure of the intestinal microbiota, including Lactobacillus, Bifidobacteria, Enterococcus, coliforms and Enterobacteriaceae, was tested and compared. After consecutive administration of casein GMP for 15 days, numbers of Lactobacillus and Bifidobacteria increased significantly (P<0.01), numbers of Enterobacteriaceae and Coliforms decreased significantly (P<0.05) while no significant changes were observed for Enterococcus. The detection limits of FISH technique were significantly lower (P<0.01) than the traditional culture method. These results suggested that consumption of casein GMP had a prebiotic effect on male BALB/c mice. Casein GMP helped establish a healthier intestinal microbiota. Additionally, FISH was proved to be a rapid and relatively low-cost detection method that can be used to further our understanding of human intestinal microbiota.