Objective: Fatty acids are crucial for cell survival, however, their over-accumulation triggers lipotoxicity that leads to cell death. It has been known oleic acid (OA) to be less toxic than palmitic acid (PA) and to attenuate PA-induced lipotoxicity. The mechanisms are not fully understood. We investigated the protective role of OA on PA-induced lipotoxicity in HepG2 cells. Methods: HepG2 cells were treated with different concentration of PA and/or OA conjugated with 10% BSA. After 24 h, cell viability and triglycerides accumulation were determined by MTT assay and Oil Red O staining, respectively. Protein expressions were detected by Western Blotting. In order to investigate the role of JNK or AKT in lipotoxicity, cells were pre-treated with JNK inhibitor (SP600125), AKT inhibitor (Wortmannin), AKT activator (GW9508) for 1h and co-treated with PA and/or OA for 24h. Data were represented by mean ± SD. Differences between the means were calculated using one-way analysis of variance (ANOVA) with a Tukey's Multiple Comparison Test (P < 0.05). Results: Intra-cellular triglycerides accumulation of two fatty acids did not differ although PA was more toxic to HepG2 cells than OA. PA, but not OA, induced lipotoxicity in JNK-dependent manner. Apoptotic proteins: P53 was increased, Bcl-2 was decreased in cells treated with PA. But OA did not activate them. OA treatment or SP600125 reduced PA-induced JNK activation and cell death. OA or GW9508 increased AKT phosphorylation and decreased PA-induced JNK activation. However, OA with Wortmannin did not affect PA-induced JNK activation. Conclusion: Despite equal cellular steatosis, PA causes JNK activation leading to lipotoxicity and OA reduces it by AKT activation in HepG2 cells.