Background: Reduction of pancreatic beta-cells mass, major secondary to increased beta-cells apoptosis, is increasingly recognized as one of the main contributing factors to the pathogenesis of type 2 diabetes (T2D), and saturated free fatty acid palmitate has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting beta-cells apoptosis. Recent literature suggests that valproate, a diffusely prescribed drug in the treatment of epilepsy and bipolar disorder, can inhibit glycogen synthase kinase-3 beta (GSK-3 beta) activity and has cytoprotective effects in neuronal cells and HepG2 cells. Thus, we hypothesized that valproate may protect INS-1 beta-cells from palmitate-induced apoptosis via inhibiting GSK-3 beta. Results: Valproate pretreatment remarkable prevented palmitate-mediated cytotoxicity and apoptosis (lipotoxicity) as well as ER distension. Furthermore, palmitate triggered ER stress as evidenced by increased mRNA levels of C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) in a time-dependent fashion. However, valproate not only reduced the mRNA and protein expression of CHOP but also inhibited GSK-3 beta and caspase-3 activity induced by palmitate, whereas, the mRNA expression of ATF4 was not affected. Interestingly, TDZD-8, a specific GSK-3 beta inhibitor, also showed the similar effect on lipotoxicity and ER stress as valproate in INS-1 cells. Finally, compared with CHOP knockdown, valproate displayed better cytoprotection against palmitate. Conclusions: Valproate may protect beta-cells from palmitate-induced apoptosis and ER stress via GSK-3 beta inhibition, independent of ATF4/CHOP pathway. Besides, GSK-3 beta, rather than CHOP, may be a more promising therapeutic target for T2D.