Wistar rats were fed with high fat diet to induce the diet-induced obesity model (DIO), and then gavaged with grape seed proanthocyanidins (GSP) of different doses. Culture-independent methods such as denaturing gradient gel electrophoresis (DGGE) and real-time fluorescent quantitative polymerase chain reaction (real-time PCR) were applied, coupled with multivariate analysis, to explore the effect of GSP on the microbiota of DIO rats. Results showed that GSP treatment (100mg/(kg·bw·d)) could inhibit high-fat induced obesity, and its gut microbiota profile was separated from that of the model group. Results also suggested that GSP-treated groups had the lower Firmicutes content and higher Bacteroidetes content and the ratio of Firmicutes to Bacteroidetes increased in this group. Real-time-PCR results indicated that GSP promoted the proliferation of Bacteriodes spp. and inhibited that of Clostridium leptum. The key genera the GSP might have an effect on were as follows:Blautia, Bacteroides, Lactobacillus, Anaerostipes, Clostridium, Anaerofilum.