Colorectal cancer (CRC) is a malignant tumor with high incidence and bad prognosis. Therapies, which are more safe and effective, are urgently needed. Trypsin is proved to be crucial to cancer proliferation and migration, therefore, it is possible to control cancers by modulating its activity. Fisetin is a flavone with trypsin inhibition properties that was screened from more than 45 compounds derived from traditional Chinese medicine (TCM). However, the effects and mechanisms of fisetin on CRC have not been well investigated. In this study, we evaluated the effects of fisetin on 2 different CRC cell lines. Fisetin remarkably inhibited CRC cell proliferation and migration, as well as induced cell apoptosis and Go/G1 phase arrest in a dose-dependent manner. Mechanistic studies revealed that these effects were mediated partially through signaling pathways involving cell cycle regulators p21, p27, cyclinD1, and NF kappa B (NF-κB) p65. Administration of fisetin also significantly suppressed the tumor growth in tumor-bearing NOD/Shi-scid-IL2R gamma (null) (NOG) mice that had been inoculated with human HCT116 cells. Fisetin at the given dosage did not induce significant acute or chronic toxicity in rats. These data provide a potential therapeutic strategy for CRC.