Abstract: In this study, the effects of honokiol (HN) treatment for 24 h on lipid synthesis was examined in HepG2 cells. The parameters include intracellular lipid droplet and the expression of SREBP-1c and PNPLA3, glucose uptake, and oxidative stress including the expression of CYP2E1 and CYP4A in normal, TO901317 (TO)- and oleic acid (OA)-treated HepG2 cells. The lipid droplets were detected by oil red O staining. The glucose uptake was measured by fluorescence spectrophotometry using2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose, 2-NBDG as probe. The expression levels of target genes were detected by quantitative PCR and Western blot. The results showed that:① TO (5 μmol·L⁻¹) and OA (0.5 mmol·L⁻¹) treatment increased the levels of intracellular lipid accumulation and the mRNA and protein expression of SREBP-1c and PNPLA3. After HN (10, 20, 40 μmol·L⁻¹) treatment for 24 h, the lipid accumulation and the expression of SREBP-1c and PNPLA3 were all decreased in the tested cells. ② OA treatment significantly suppressed glucose uptake, while HN treatment dose-dependently increased the glucose uptake in OA-treated cells. ③ Compared with control group, CYP2E1 protein level significantly decreased in the three tested cells, and CYP4A protein level significantly decreased only in OA-treated cells following HN treatment. The above results suggest that HN may attenuate lipid accumulation by suppressing the expression of SREBP-1c and PNPLA3, and reduce lipid peroxidation and insulin resistance by down-regulation of the protein levels of CYP2E1 and CYP4A in HepG2 cells with steatosis.