Upstream transcription factor 1 (USF1) regulates the expression of many genes involved in lipid and glucose metabolism, among them genes regulating lipolysis. USF1 specifically regulates the expression of the hormone-sensitive lipase gene (HSL) in adipocytes and the hepatic lipase gene (LIPC) in the liver, which was found to be involved in liver fat accumulation. The usf1s1 C > T and usf1s2 G > A single-nucleotide polymorphisms (SNPs) in USF1 are associated with increased in vitro catecholamine-induced lipolysis in adipocytes. We investigated first whether SNPs in USF1 affect the lipolysis-suppressing action of insulin in vivo, and second, whether they interact with the -60C > G SNP in HSL on lipolysis and the -514C > T SNP in LIPC on liver fat. The usf1s1 C > T and usf1s2 G > A SNPs, together with the SNPs in HSL and LIPC, were determined in 407 Caucasians. Lipolysis was estimated as a change in free fatty acid (FFA) levels from baseline to 2 h of a 75-g oral glucose tolerance test (OGTT). Fifty-four subjects had data from a euglycemic hyperinsulinemic clamp with calculation of antilipolytic insulin sensitivity. Subjects carrying the minor alleles (T of usf1s1 and A of usf1s2) had lower 2 h FFA (p = 0.01) and a larger decrease in FFA concentrations during the OGTT (p = 0.02). Antilipolytic insulin sensitivity was higher in these individuals (p = 0.03). No interaction of the usf1s1 C > T and usf1s2 G > A SNPs with the -60C > G SNP in HSL on antilipolytic insulin sensitivity was detected. Liver fat, measured by (1)H magnetic resonance spectroscopy, was elevated only in subjects who were both homozygous for the major alleles of usf1s1 and usf1s2 and carriers of the T allele of the -514C > T SNP in LIPC (p = 0.01). In conclusion, subjects carrying the T allele of SNP usf1s1 and the A allele of SNP usf1s2 have a higher antilipolytic insulin sensitivity. Moreover, both SNPs may interact with the -514C > T SNP in LIPC to determine liver fat.