Objective— Hepatic ATP-binding cassette transporter A1 (ABCA1) plays the major role in maintaining plasma high-density lipoprotein levels by producing cholesterol-accepting nascent high-density lipoprotein, whereas peripheral ABCA1 is responsible for releasing cellular cholesterol. We previously reported that in rodents, cholesterol depletion reduces ABCA1 expression in peripheral but not hepatic cells by increasing a liver-specific ABCA1 transcript via the sterol regulatory element-binding protein-2 system. However, the regulatory element is not conserved in humans. Here we investigated the mechanism of sterol-regulated human hepatic ABCA1 gene expression. Methods and Results— ABCA1 mRNA variant type L3 is a novel and human-liver-specific transcript accounting for ≈25% of total ABCA1 mRNA in the liver and is induced by cellular cholesterol depletion. Specific knockdown or forced expression revealed that type L3 produces functional ABCA1 protein in cholesterol efflux. We identified a regulatory enhancer element for L3 expression lying within intron 3 of the human ABCA1 gene, to which hepatocyte nuclear factor (HNF) 4α binds in response to cholesterol depletion. HNF4α knockdown abolished induction of liver-specific L3 and L2b transcripts (and consequently the liver-type response of ABCA1 expression to cellular cholesterol status) and diminished cholesterol efflux activity. Conclusion— These findings indicate that HNF4α regulates human hepatic ABCA1 expression in response to cholesterol depletion.