Lipid accumulation in podocytes can lead to the destruction of cellular morphology, in addition to cell dysfunction and apoptosis, which is a key factor in the progression of chronic kidney disease (CKD). Berberine (BBR) is an isoquinoline alkaloid extracted from medicinal plants such as Coptis chinensis, which has been reported to have a lipid-lowering effect and prevent CKD progression. Therefore, the present study aimed to investigate the effect of BBR on palmitic acid (PA)-induced podocyte apoptosis and its specific mechanism using an in vitro model. Cell death was measured using the Cell Counting Kit-8 colorimetric assay. Cell apoptotic rate was assessed by flow cytometry. The expression of endoplasmic reticulum (ER) stress- and apoptosis-related proteins was detected by western blotting or immunofluorescence. Reactive oxygen species (ROS) were evaluated by 2 ' ,7 ' -dichlorofluorescein diacetate fluorescence staining. The results of the present study revealed that BBR treatment decreased PA-induced podocyte apoptosis. In addition, 4-phenylbutyric acid significantly reduced PA-induced cell apoptosis and the expression of ER stress-related proteins, which indicated that ER stress was involved in PA-induced podocyte apoptosis. In addition, N-acetylcysteine inhibited PA-induced excessive ROS production, ER stress and cell apoptosis of podocytes. BBR also significantly reduced PA-induced ROS production and ER stress in podocytes. These results suggested that PA mediated podocyte apoptosis through enhancing ER stress and the production of ROS. In conclusion, BBR may protect against PA-induced podocyte apoptosis, and suppression of ROS-dependent ER stress may be the key mechanism underlying the protective effects of BBR.