Objective To establish a conditional knockout mouse model of transcription factor carbohydrate response element-binding protein (ChREBP), so as to provide a technical approach for exploring the biological function of ChREBP in vivo. Methods and results Using Cre/loxP gene targeting strategy, we constructed the gene targeting vector and franked the 8^th exon of ChREBP gene with loxP site in embryonic stem cells (ES cells) via homologous recombination. We microinjected the recombined ES clones into blastocysts and implanted into the uterus of pseudopregnant mice to obtain the chimeric mice and ChREBP^flox/+ mice. By crossbreeding ChREBP^flox/+ mice with Alb-Cre mice, we generated liver-specific ChREBP knockout mice. Conclusion The generation of ChREBP conditional knockout mouse model provides an important means to reveal the physiological function and pathological significance of ChREBP in different tissues.