The effects of banana resistant starch on the alteration of gut microbiota in Human Flora-Associated (HFA) mouse model fed on a high fat diet, were investigated. Thirty HFA mice were randomly assigned into three groups (n = 10/group), which were fed a normal diet, high fat diet, and high fat diet + resistant starch, respectively. DNA was extracted from the fresh feces of all mice, which were collected prior to the beginning of the experiment (0th week) and after eight weeks (8th week). The primer-specific amplification products using the V6-V8 region of 16S rDNA were compared and differences in the profile of gut microbiota among the resistant starch, high fat diet, and normal diet groups were tested using polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE). Richness, diversity index, and evenness value of gut microbiota were not significantly different between the three groups at 0th week (p > 0.05). Flora similarity between the mice and the human feces donor was 36%, but the flora similarity between the three mice groups ranged from 60% to 93%. Principal component analysis (PCA) showed that the data points of all three groups were clustered together, indicating no difference between the three groups before intervention and that the HFA mouse model was suitable for the investigation of resistant starch on gut microbiota. Richness and diversity index values of gut microbiota between the resistant starch group and the other two groups 8 weeks after the dietary intervention were significantly different (p < 0.01). PCA of the mouse intestinal bacterial community showed that data points were distributed within different zones after the dietary interventions. In conclusion, resistant starch has a huge influence on the gut bacterial community structure of HFA mice fed on a high fat diet.