Recent studies highlight the importance of lipotoxic damage in aortic cells as the major pathogenetic contributor of atherosclerotic disease. Since the STE20-type kinase STK25 has been shown to exacerbate ectopic lipid storage and associated cell injury in several metabolic organs, we here investigated its role in the main cell types of vasculature. We depleted STK25 by small interfering RNA in human aortic endothelial and smooth muscle cells exposed to oleic acid and oxidized LDL. In both cell types, the silencing of STK25 reduced lipid accumulation and suppressed activation of inflammatory and fibrotic pathways as well as lowered oxidative and endoplasmic reticulum stress. Notably, in smooth muscle cells, STK25 inactivation hindered the shift from a contractile to a synthetic phenotype. Together, we provide the first evidence that antagonizing STK25 signaling in human aortic endothelial and smooth muscle cells is atheroprotective, highlighting this kinase as a new potential therapeutic target for atherosclerotic disease.