Treatment of colorectal cancer (CRC) remains a challenge because of the lack of effective early treatment strategies and high incidence of relapse. 5-Fluorouracil (5-FU) is a typical CRC treatment. Bromosporine is an innovative bromodomain and extraterminal domain (BET) inhibitor. We investigated if CRC could be targeted by the combination of 5-FU and bromosporine in a synergistic manner in vivo and in vitro. Our findings shown that the combination treatment inhibits cell viability, formation of colonies, increased apoptosis and cell cycle arrest at G0-G1. In addition, the expression level of BRD4 was high in HCT116 cells exposed to 5-FU that showed lower apoptosis against the parental cells. Moreover, the 5-FU-resistance was reversed significantly by BRD4 knockdown or inhibition. The drug combination showed increased activity against tumor than individual drug exposure in the xenograft model. In conclusion, this work serves as a basic clinical evaluation of 5-FU and bromosporine as an effective therapeutic approach for CRC. (C) 2019 Elsevier Inc. All rights reserved.