Objective To construct recombinant adenovirus expressing mouse ChREBP-α, and examine the effect of ChREBP-α overexpression on lipid synthesis in mouse primary hepatocytes. Methods The mouse ChREBP-α cDNA was subcloned into pShuttle-CMV vector, and the product was transformed into E. coli strain BJ5183 for homologous recombination with pAdEasy-1．The resultant recombinant vector was transfected into 293A cells for viral package. Mouse primary hepatocytes were infected with adenoviruses Ad-ChREBP-α, and gene expression was analyzed at mRNA and protein levels by real-time PCR and Western blotting analsysis. The expression levels of ChREBP target gene LPK were measured at mRNA level by real-time PCR, and the fatty acid synthesis rate was determined by \[¹⁴C\]-acetate incorporation. Results We successfully constructed recombinant adenoviruses expressing mouse ChREBP-α. Adenovirus-mediated overexpression of ChREBP-α markedly increased LPK mRNA expression and fatty acid synthesis in primary hepatocytes. Conclusion We have successfully constructed recombinant adenoviruses expressing mouse ChREBP-α, which is biologically active and can overexpress ChREBP-α in primary hepatocytes. Overexpression of ChREBP-α can enhance lipid synthesis.