Emodin is the main toxic component in Chinese medicinal herbs such as rhubarb. Our previous studies demonstrated that genetic polymorphisms of UDP-glucuronosyltransferase 2B7 (UGT2B7) had an effect on the glucuronidation and detoxification of emodin. This study aimed to reveal the transcriptional regulation mechanism of UGT2B7 on emodin glucuronidation and its effect on toxicity. Emodin glucuronic activity and genome and transcriptome data were obtained from 36 clinical human kidney tissues. The genome-wide association studies (GWAS) identified that four single nucleotide polymorphisms (SNPs) (rs6093966, rs2868094, rs2071197, and rs6073433), which were located on the hepatocyte nuclear factor 4α (HNF4A) gene, were significantly associated with the emodin glucuronidation (p < 0.05). Notably, rs2071197 was significantly associated with the gene expression of HNF4A and UGT2B7 and the glucuronidation of emodin. The gene expression of HNF4A showed a high correlation with UGT2B7 (R2 = 0.721, p = 5.83 × 10-11). The luciferase activity was increased 7.68-fold in 293T cells and 2.03-fold in HepG2 cells, confirming a significant transcriptional activation of UGT2B7 promoter by HNF4A. The knockdown of HNF4A in HepG2 cells (36.6%) led to a significant decrease of UGT2B7 (19.8%) and higher cytotoxicity (p < 0.05). The overexpression of HNF4A in HepG2 cells (31.2%) led to a significant increase of UGT2B7 (24.4%) and improved cell viability (p < 0.05). Besides, HNF4A and UGT2B7 were both decreased in HepG2 cells and rats after treatment with emodin. In conclusion, emodin used long term or in high doses could inhibit the expression of HNF4A, thereby reducing the expression of UGT2B7 and causing hepatotoxicity.