[Background] Dietary fiber has been regarded as the seventh nutrients and can be utilized by the microbes in the hindgut of monogastric animals. [Objective] The present study was conducted to investigate the influence of oat β-glucan (a typical soluble dietary fiber) and microcrystalline cellulose (MCC, an insoluble dietary fiber) on the structure and composition of colonic bacteria in BALB/c mice. Results of current study can provide a reference for the formulating of dietary fiber containing feed in animal production and the rational utilization of different types of dietary fibers in human food. [Methods] A total of 27 healthy male BALB/c mice (18.13±0.95 g) at the age of six weeks were selected and randomly allocated to three groups. Mice in the three groups were fed diet containing 20% MCC (the purity≥99%, M), 28% oat β-glucan (the purity is 70%, G), and control diet without fibrous supplement, respectively. The experiment lasted for 21 days. At the end of the experiment, three mice from each group were sacrificed and the colonic digesta of each mouse was collected. The bacterial community of the digesta samples from the three groups were compared using PCR-DGGE (Polymerase chain reaction-denaturing gradient gel electrophoresis) and high-throughput sequencing methods. [Results] PCR-DGGE analysis showed significant differences on the richness and Shannon index of the three groups, of which group G presented lower than groups M and control (P=0.027, 0.035). The cluster analysis showed that there were two samples of each group clustered into separate clade and the similarities of bands for group G, M and control were 71%, 55% and 67%, respectively. The results of high-throughput sequencing showed a significant difference on the bacterial Shannon index and β-diversity among the three groups (P=0.047, 0.035). In all samples, Bacteroidetes, Firmicutes and Proteobacteria were identified as the most three predominant phyla, comprising 95.9% to 99.4% of the total reads. Compared to control group, the relative abundance of phylum Bacteroidetes in group G showed a 26.78% increase, while it showed a 15.62% decrease in group M of those genera in Bacteroidetes, an unclassified genus belonging to family S27_4 and Bacteroides made a maximum contribution to the change of this phylum (P=0.099, 0.051). On the other hand, the relative abundance of Firmicutes in group G showed a 28.99% decrease than control group, while it showed a 15.82% increase in group M, and this change was found mainly due to the change of relative abundance of order Clostridiales, Ruminococcaceae and Lactobacillus (P=0.027, 0.061 and 0.079, respectively). [Conclusion] Therefore, both of the two dietary fibers influenced the bacterial community in the colon of BALB/c mice. The supplement of high-level oat β-glucan in the diet decreased the bacterial diversity in the colon of the mice. Core bacteria groups specifically utilizing the two types of dietary fibers were found in the colon of BALB/c mice. Bacteria belonging to family S27_4 may prefer to utilize plant polysaccharide, such as oat β-glucan, while some bacteria belonging to order Clostridiales may specifically use MCC.