ATP-citrate lyase (ACLY) is the major source of glucose-derived acetyl-CoA outside the mitochondria and is an attractive therapeutic target for metabolic disorders and cancer. In the cytosol, acetyl-CoA is used for lipid synthesis and is also the essential substrate for protein acetylation. In the nucleus, acetyl-CoA is used for histone acetylation, which is particularly sensitive to glucose availability and ACLY deficiency. ACLY protein is present in both the cytosol and nucleus, but the significance is unclear since acetyl-CoA can diffuse between the cytosol and nucleus through nuclear pores. In addition, the ACLY transcript has two major splice isoforms, with a cassette exon located in an intrinsically disordered region (IDR). In this thesis, I analyze the cellular regulation of ACLY regarding alternative splicing and subcellular localization.Foremost, I identify epithelial splicing regulatory factor 1 (ESRP1) as one regulator of ACLY splicing. Although isoform expression patterns differ in human tumors compared to normal tissues, no discernible differences in enzymatic activity, cellular functions, or metabolic and tumor-related phenotypes in vivo were observed as a function of ACLY splicing. Subsequently, I identified an endogenous nuclear localization sequence in the IDR of ACLY and leveraged cell lines in which ACLY is targeted to the nucleus or cytosol to investigate its compartmentalized. Overall, the data support a model in which acetyl-CoA generated in either the cytosol or nucleus can be used for processes across these compartments, but that compartmentalized ACLY allows for fine-tuning of both lipid metabolism and histone acetylation. Finally, I discuss pertinent literature on bempedoic acid (BPA), a first-in-class drug for the treatment of hypercholesterolemia and cardiac risk reduction, by which inhibition of ACLY in the liver is widely accepted as the major mechanism. However, BPA treatment also has effects on lipid metabolism that are ACLY-independent, as BPA’s structural similarity to endogenous fatty acids allows it to trigger multiple lipid-signaling pathways. I highlight additional molecular targets of BPA and structurally related drugs with the aim of providing a resource for researchers and clinicians as BPA is more widely prescribed.