ATP citrate lyase (ACLY) catalyzes the reaction: citrate + CoA + ATP → acetyl-CoA + oxaloacetate +ADP + Pi (Srere & Lipmann, 1953). Because acetyl-CoA is the precursor of de novo lipogenesis, human ACLY (hACLY) is linked to multiple diseases (Groot et al., 2003; Bauer et al., 2005). The goal of this study is to understand the catalytic and regulatory mechanism of hACLY. Crystals of a rationally designed form of the amino terminus of hACLY showed electron density of the catalytic loop for the first time. The binding modes of the inhibitors, 4R- and 4S-hydroxycitrate, were seen, and it was concluded that either the hydroxycitryl-CoA cannot be formed or cannot be cleaved. Binding assays were done to determine whether 14-3-3 protein binds directly to phosphorylated serine-455 of hACLY. The results suggest that 14-3-3 protein binds to hACLY, but not specific ally to phosphorylated Ser-455.