To understand cholesterol-mediated regulation of human fatty acid synthase promoter I, we tested various 5′-deletion constructs of promoter I-luciferase reporter gene constructs in HepG2 cells. The reporter gene constructs that contained only the Sp-1-binding site (nucleotides −82 to −74) and the two tandem sterol regulatory elements (SREs; nucleotides −63 to −46) did not respond to cholesterol. Only the reporter gene constructs containing a nuclear factor-Y (NF-Y) sequence, the CCAAT sequence (nucleotides −90 to −86), an Sp-1 sequence, and the two tandem SREs responded to cholesterol. The NF-Y-binding site, therefore, is essential for cholesterol response. Mutating the SREs or the NF-Y site and inserting 4 bp between the Sp-1- and NF-Y-binding sites both resulted in a minimal cholesterol response of the reporter genes. Electrophoretic mobility-shift assays using anti-SRE-binding protein (SREBP) and anti-NF-Ya antibodies confirmed that these SREs and the NF-Y site bind the respective factors. We also identified a second Sp-1 site located between nucleotides −40 and −30 that can substitute for the mutated Sp-1 site located between nucleotides −82 and −74. The reporter gene expression of the wild-type promoter and the Sp-1 site (nucleotides −82 to −74) mutant promoter was similar when SREBP1a [the N-terminal domain of SREBP (amino acids 1–520)] was constitutively overexpressed, suggesting that Sp-1 recruits SREBP to the SREs. Under the same conditions, an NF-Y site mutation resulted in significant loss of reporter gene expression, suggesting that NF-Y is required to activate the cholesterol response.