The acetyl-CoA carboxylase isoform ACC2 expressed in the liver generates malonyl-CoA, which primarily regulates fatty acid oxidation through inhibition of the mitochondrial carrier carnitine palmitoyl-CoA transferase-I. Activity is initiated by sterol regulatory element-binding protein-1 (SREBP-1) binding to steroid response elements SRE in ACACB gene promoter P-II. We proposed that sequence variation in the promoter might affect expression. We investigated the effect of a single-nucleotide polymorphism -368 C/T (rs16939972) in ACACB P-II on activity in transfected HepG2 hepatoma cells. The T-allele construct showed significantly lower activity than the C-allele (p = 0.016) but only in the presence of SREBP-1a overexpression. Electrophoretic mobility shift assays showed that HepG2 nuclear proteins bound specifically to both allele probes, but with higher affinity to the T-allele. We tested competition for nuclear protein binding between the T-allele probe and unlabeled probes containing consensus sequences for six candidate transcription factors plus SREBP-1a. The SREBP-1a competitor probe had no effect on the shifted complex. GATA, c-Myb, and GR competitor probes abolished the complex; however, these proteins were undetectable in mass spectrometry of gel extracts from shifted bands. In conclusion, the -368 C/T single-nucleotide polymorphism in ACACB P-II binds HepG2 nuclear proteins that affect promoter activity in an allele-specific fashion.