Hemistepsin A (HsA) is a guaianolide sesquiterpene lactone that inhibits hepatitis and liver fibrosis. We evaluated the effects of HsA on liver X receptor (LXR)-mediated hepatic lipogenesis in vitro and in vivo. Up to 10 μM, HsA did not affect the viability of HepG2 and Huh7 cells. Pretreatment with 5-10 μM HsA significantly decreased the luciferase activity of the LXR response element, which was transactivated by T0901317, GW 3965, and LXRα/retinoid X receptor α overexpression. In addition, it significantly inhibited the mRNA expression of LXRα in HepG2 and Huh7 cells. It also suppressed the expression of sterol regulatory element-binding protein-1c and lipogenic genes and reduced the triglyceride accumulation triggered by T0901317. Intraperitoneal injection of HsA (5 and 10 mg/kg) in mice significantly alleviated the T0901317-mediated increases in hepatocyte diameter and the percentage of regions in hepatic parenchyma occupied by lipid droplets. Furthermore, HsA significantly attenuated hepatic triglyceride accumulation by restoring the impaired expression of LXRα-dependent lipogenic genes caused by T0901317. Therefore, based on its inhibition of the LXRα-dependent signaling pathway, HsA has prophylactic potential for steatosis. [BMB Reports 2021; 54(2): 106-111].