Acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) is important for cholesterol ester synthesis and secretion. A previous study revealed that ACAT2 gene promoter activity was upregulated by hepatocyte nuclear factor 4α (HNF4α) through two sites around -247 and -311 of ACAT2 gene promoter. Here, we identified two novel cis-elements, site I (-1006 to -898) and site II (-38 to -29), which are important for HNF4α effect. In HepG2 cells, mutation of site I decreased ACAT2 gene promoter activity to one-fifth of that of the wild type, while mutation of site II reduced promoter activity to less than one-tenth of that of the wild type. In 293T cells, mutation of these two cis-elements profoundly impaired the HNF4α induction effect. When either of these two elements was inserted into pGL3-promoter, HNF4α induced promoter activity through the inserted element, while mutation of the element impaired HNF4α induction effect. In electrophoretic mobility shift assay and chromatin immunoprecipitation experiment, HNF4α bound to these two elements. Thus, the two cis-elements are important for HNF4α effect on ACAT2 gene transcription. We also showed that HNF4α positively regulates ACAT2 gene expression at mRNA level. Overexpression of HNF4α increased ACAT2 expression, whereas knockdown of HNF4α decreased ACAT2 expression. Peroxisome proliferator-activated receptor gamma coactivator 1α (PCG1α), a coactivator of HNF4α, increased ACAT2 expression, while small heterodimer partner (SHP), a corepressor of HNF4α, decreased ACAT2 expression. These results provide more insights into transcriptional regulation of ACAT2 expression.