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 ¹H 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.