ATP-citrate lyase (ACL), one of the lipogenic enzymes, catalyses the formation of acetyl-coenzyme A (CoA) involved in the synthesis of fatty acid and cholesterol. In pig, very little is known about the ACL gene. In this work, the mRNA differential display technique was used to analyse the differences in gene expression between Meishan and Large White pigs and the F1 hybrids of both direct and reciprocal crosses. Our results show that among the differentially expressed genes ACL is up-regulated in the backfat of the F1 hybrids. After cloning and analysing the full-length cDNA and the 870 bp 50-flanking sequence of the porcine ACL gene, a C/T mutation at position -97 bp upstream of the transcription site was detected. Luciferase activity detection showed that this mutation changed the transcriptional activity. In F1 hybrids, the heterozygous genotype CT was more frequent than the homozygous genotypes CC and TT. Real-time PCR analysis showed that in Meishan pigs, ACL mRNA expression was more abundant in individuals with genotype CT than in those with genotype CC or TT or in Large White pigs. These results indicate that the C/T mutation affects ACL mRNA expression, probably via the activator protein 2.