Hyperlipidemia is a major risk factor for cardiovascular diseases, emphasizing the need for effective regulation of cholesterol homeostasis. Low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) are key regulators of hepatic cholesterol clearance. Sesamin, a lignan abundant in sesame seed oil, has been reported to exhibit antioxidant, anti-inflammatory, and anticancer properties. However, the mechanisms underlying its lipid-lowering effects remain unclear. This study aimed to explore the mechanisms by which sesamin regulates cholesterol homeostasis. Sesamin treatment significantly decreased PCSK9 expression at both mRNA and protein levels while increasing LDLR expression. To determine whether this effect involves direct binding, the cellular thermal shift assay (CETSA) and in vitro binding analysis were performed. Results showed that sesamin increased the thermal stability of PCSK9, and binding assays confirmed that sesamin inhibited the PCSK9-LDLR interaction, indicating that sesamin acts as a direct blocker of PCSK9. Furthermore, siRNA-mediated knockdown experiments demonstrated that sesamin regulates PCSK9 and LDLR expression via transcription factors such as HNF1α and SREBP2. In addition, sesamin increased SIRT1 and AMPK phosphorylation, suggesting involvement of these signaling pathways. Collectively, these findings demonstrate that sesamin improves cholesterol homeostasis by directly inhibiting PCSK9-LDLR interaction and transcriptionally regulating their expression.