Considering the absence of prior studies on the cholesterol metabolism-improving effects of eugeniin, the present investigation aimed to explore the potential impact of eugeniin on cholesterol metabolism. This study sought to elucidate the molecular mechanisms involved in this process using HepG2 and Caco-2 cells treated with 5 µm eugeniin. The intracellular cholesterol levels in HepG2 and Caco-2 cells were significantly decreased in the 24-h eugeniin-treated group. The protein and messenger ribonucleic acid (mRNA) levels of the low-density lipoprotein receptor (LDLR) were increased, while 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase protein and mRNA levels were decreased in HepG2 cells 6h of the eugeniin-treated group. Additionally, LDLR protein and mRNA levels were increased in HepG2 cells after 24h of eugeniin treatment. In Caco-2, the protein and mRNA levels of ATP-binding cassette transporter 1 were increased after 24h eugeniin treatment. This novel finding indicates that eugeniin improves cholesterol metabolism in human cell cultures.