Food supplements can modulate the composition of human intestinal microflora due to the potential capacity of improving the host health. Chitosan (CS) possesses diverse biological and pharmacological effects and has been used as a new source of prebiotic. In this study, ERIC (Enterobacterial Repetitive Intergenic Consensus)-PCR and viable counts methods were combined to explore the prebiotic-like effects of CS on the intestinal microflora in mice. Mice were divided into 3 groups randomly, and treated with water, 1g/kg of CS, 0.25g/kg of CS, respectively for 24 days. Subsequently, they were treated with levofloxacin (65mg/kg) for 6 days. Viable counts method indicated the growth of Lactobacillus was promoted with CS treatment while at the same time Enterobacteria and Enterococcus were inhibited. ERIC-PCR fingerprint, UPGMA dendrogram, PCA and diversity analysis showed that the intestinal microflora composition was changed with antibiotics treatment, and that samples were significantly separated from those of the control and long-term CS-treated groups. Together, our results demonstrated that CS could be regarded as a potential food supplement for protecting intestinal microflora and regulate imbalance.