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The gut microbiota has been shown to be involved in host energy homeostasis and diet-induced metabolic disorders. To gain insight into the relationships among diet, microbiota and the host, we evaluated the effects of a high-fat (HF) diet on the gut bacterial community in weaning mice. C57BL/6 mice were fed either a control diet or a diet enriched with soy oil for 1 and 2 weeks. Administration of the HF diet caused an increase in plasma total cholesterol levels, while no significant differences in body weight gain were observed between the two diets. Denaturing gradient gel electrophoresis (DGGE) profiles indicated considerable variations in the caecal microbial communities of mice on the HF diet, as compared with controls. Two DGGE bands with reduced intensities in HF-fed mice were identified as representing Lactobacillus gasseri and an uncultured Bacteroides species, whereas a band of increased intensity was identified as representing a Clostridium populeti-related species upon sequencing. Quantitative real-time PCR confirmed a statistically significant 1-log decrease in L. gasseri cell numbers after HF feeding, and revealed a significantly lower level of Bifidobacterium spp. in the control groups after 1 and 2 weeks compared with that in the HF groups. These alterations of intestinal microbiota were not associated with caecum inflammation, as assessed by histological analysis. The observed shifts of specific bacterial populations within the gut may represent an early consequence of increased dietary fat.