Gemfibrozil (GEM) is a phenoxy aromatic acid-based lipid-lowering drug. It activates peroxisome proliferator-activated receptor alpha (PPAR-α), which leads to altered lipid metabolism and lowers serum triglyceride levels by modulating lipoprotein lipase. However, the action of the mode of GEM is still unclear. Herein, the model organism Saccharomyces cerevisiae was applied to explore the molecular mechanism of GEM regulating lipid metabolism. The results showed that the triacylglycerol (TAG) content and the number of lipid droplets of yeast increased significantly after GEM treatment in the wild-type BY4741. Screening of mutations related to lipid metabolism pathways (PAH1, DGK1, TGL3, TGL4, LRO1, ARE1, ARE2, and DGA1) showed that dgk1Δ had no change in lipid accumulation under GEM. In the wild type, GEM inhibited the expression of DGK1, resulting in a significant decrease in the contents of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS)) and neutral lipids (TAG and diacylglycerol (DAG)). However, their abundances could not be changed in dgk1Δ after the treatment with GEM Luciferase assay further showed that DGK1 may be a target of GEM to induce lipid accumulation via TUP1/CYC8, which could act on the DGK1 promoter-TATA highly conserved element (-400 bp - 200 bp). Altogether, the effect of GEM on lipid metabolism was associated with the upregulation of TUP1/CYC8, leading to a decrease in the expression of DGK1, thereby increasing the TAG content in yeast cells. It is expected that the data will help to clarify the molecular mechanism of GEM regulating lipid metabolism in humans.