To investigate the modulatory effect of oolong tea polyphenols (OTP) on intestinal microbiota, OTP was prepared by column chromatography and its influence on the gut flora structure was analyzed by high-throughput sequencing with a human flora-associated high fat diet (HFD) induced obesity mouse model. We observed a robust increase in bacterial biodiversity and the abundance of genera known to be butyrate- and acetate-producing bacteria. A large increase in Bacteroidetes with a decrease in Firmicutes was observed after the administration of OTP for 4 weeks, and the corresponding decrease in the Firmicutes/Bacteroidetes ratio reflected the positive modulatory effect of OTP on the intestinal microbiota. In addition, KEGG pathways for the biosynthesis of amino acids, carbon metabolism, and the ribosome were among the most differentially expressed genes after OTP intervention. The current study revealed that OTP rich in tea catechins, especially O-methylated derivatives, may have prebiotic-like activity and can be used as a functional food component with potential therapeutic utility to prevent obesity-related metabolic disorders by manipulating the intestinal microbiota.