Background: Berberine (BBR) has been extensively reported to inhibit colorectal cancer (CRC) development, though its bioavailability is poor. Nowadays, an increasing number of studies have shown that BBR significantly accumulates in the intestines and could regulate gut microbiota in obesity. The purpose of this study was to further explore the effects of BBR on gut microbiota in Apc ^min/+ mice receiving a high fat diet (HFD). Methods: Apc ^min/+ mice received either HFD alone or HFD and BBR for 12 weeks. The intestinal tissues were collected to evaluate the efficiency of BBR on neoplasm development by hematoxylin and eosin staining. Meanwhile, immunohistochemistry was conducted to investigate the effects of BBR on cyclin D1 and β-catenin in colon tissues. Fecal samples were subjected to 16S rRNA sequencing. Results: BBR significantly reduced intestinal tumor development and altered the structure of gut microbiota in Apc ^min/+ mice fed with an HFD. At the phylum level, it was able to significantly inhibit the increase in Verrucomicrobia. At the genus level, it was able to suppress Akkermansia and elevate some short chain fat acid (SCFA)-producing bacteria. Conclusions: BBR significantly alleviated the development of CRC in Apc ^min/+ mice fed with HFD and restored the enteric microbiome community.