Neohesperidin (NHP), derived from citrus fruits, has attracted considerable interest due to its preventative and therapeutic effects on numerous diseases. However, little progress has been made in determining the exact function of NHP on tumorigenesis. In the current study, we found that NHP inhibited colorectal tumorigenesis in the APC min/+ transgenic mouse model, as well as induced apoptosis and blocked angiogenesis in vivo. Our in-cell study suggested that this tumorigenic preventative effect of NHP is not due to the direct impact on tumor cells. Intriguingly, by utilizing 16 s rRNA gene-based microbiota sequencing, the relative abundance of Bacteroidetes was decreased, while Firmicutes and Proteobacteria were increased in the presence of NHP. Additionally, the fecal microbiota transplantation experiment further revealed that feeding with fecal of NHP-treated mice induced considerable inhibition of tumorigenesis, which indicates that the alteration of gut microbiota is responsible for NHP-mediated prevention of colorectal tumorigenesis. Thus, our study not only suggests the efficacy of NHP as a potent natural product for preventing colorectal cancer but also proposes a compelling model to connect the gut microbiota to the preventative effect of NHP on tumorigenesis.