Bromodoamin and extraterminal (BET) protein inhibitors are a novel class of cancer therapeutics. Here we aim to investigate the efficacy and mechanism of JQ-1, a potent BET inhibitor, in colon cancer therapy. JQ-1 was used to treat SW480 colon cancer mouse xenografts. The tumor size and mouse survival were recorded. Cell apoptosis was evaluated by Annex V-FIC/PI flow cytometry. ChIP-q-PCR analysis was used to assess the H3K27 trimethylation (H3K27m3) of the p16 promoter. Wnt signaling was evaluated by Nkd2 and β-catenin levels. RT-PCR was used to evaluate the level of miR-21. MiR-21 was overexpressed with a lentiviral system and was used to evaluate the relationship between miR-21 and JQ-1. JQ-1 significantly reduced tumor growth, improved mouse survival, and induced apoptosis. JQ-1 epigenetically inhibited the H3K27me3 promoter activity, promoting p16 expression. Nkd2 and β-catenin were upregulated and downregulated by JQ-1, respectively. MiR-21 was downregulated by JQ-1. MiR-21 overexpression compensated for proliferation inhibition by JQ-1. Nkd2 levels were also downregulated by miR-21 overexpression. JQ-1 is effective in inhibiting colon cancer. We revealed that the mechanism of JQ-1 action is associated with its regulation of Wnt/β-catenin signaling and miR-21 levels.