DNA-binding protein A (dbpA) is reported to be upregulated in many cancers and associated with tumor progress. The present study aimed to investigate the role of dbpA in 5-Fluorouracil (5-FU)-resistant and Oxaliplatin (L-OHP)-resistant colorectal cancer (CRC) cells. We found that 5-FU and L-OPH treatment promoted the expression of dbpA. Enhanced dbpA promoted the drug resistance of SW620 cells to 5-FU and L-OHP. DbpA knockdown inhibited cell proliferation, induced cell apoptosis and cell cycle arrested in SW620/5-FU and SW620/L-OHP cells. Besides, dbpA shRNA enhanced the cytotoxicity of 5-FU and L-OHP to SW620/5-FU and SW620/L-OHP cells. Meanwhile, dbpA shRNA inhibited the activation of the Wnt/β-catenin pathway that induced by 5-FU stimulation in SW620/5-FU cells. Activation of the Wnt/β-catenin pathway or overexpression of Chk1 abrogated the promoting effect of dbpA downregulation on 5-FU sensitivity of CRC cells. Importantly, downregulation of dbpA suppressed tumor growth and promoted CRC cells sensitivity to 5-FU in vivo. Our study indicated that knockdown of dbpA enhanced the sensitivity of colorectal cancer cells to 5-FU via Wnt/β-catenin/Chk1 pathway, and DbpA may be a potential therapeutic target to sensitize drug resistance colorectal cancer to 5-FU and L-OHP. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.