Objective To investigate the effects of different doses Alpha-Linolenic acid (ALA) on the expressions of fatty acid synthesis-related genes in HepG2 cells. Methods HepG2 cells were divided into two groups, normal control group (NC) and high fat group (HF) in which cells were firstly cultured for 36h in the medium contained 0.5mmol/L stearic acid. Real-time quantitative PCR was taken to detect mRNA expression of genes SREBP1C, FAS and ACC which are related to fatty synthesis. While there are significant differences in fatty synthesis, 10%, 20%, 50%, 70% and 100% ALA took the place of stearic acid, 36h later, real-time quantitative PCR and Western blotting were taken to detect mRNA and protein expression of genes related to fatty synthesis. Results SREBP1C mRNA expression of ALA substitution groups were significantly lower than the high-fat group (P< 0.001).The FAS of 0.5 mmol/L ALA group and 0.35 mmol/L ALA group were significantly lower than the high-fat group (P < 0.001). ACC genes mRNA level was not significantly different from high-fat group. SREBP1C and FAS protein expression were significantly lower than the high-fat group, but ACC showed no significant difference. Conclusions Saturated fatty acids promote hepatocyte fatty acid synthesis, ALA abates fatty acid synthesis by inhibiting FAS and SREBP1C gene expression.