Amiodarone is a highly effective anti-arrhythmic drug, but the clinical application of amiodarone is limited by serious adverse effects including ocular toxicity. The aim of this study was to evaluate the protective effect of anti-malarial drug artemisinin against amiodarone-induced oxidative damage, and explore the underlying molecular mechanisms in human retinal pigment epithelial cells. The cell viability was measured by MTT assay, intracellular reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) were assessed by using DCFH-DA probe and JC-1 probe respectively, cell apoptosis was analyzed by Flow Cytometry. The protein level of phosphorylated AMPK (p-AMPK), CaMMK2, LKB1, Nrf2, SOD1 and GAPDH were detected by Western blot. Our results revealed that artemisinin could attenuate amiodarone-induced cell viability decrease, intracellular ROS level increase and mitochondrial membrane potential (△ψm) depolarization in D407 cells. The artemisinin also upregulated the phosphorylation level of AMPK protein, and expression level of CaMMK2, Nrf2, and SOD1 proteins in D407 cells. However, the AMPK inhibitor Compound C reversed the protective effect of artemisinin against amiodarone-induced cell viability decrease and cell apoptosis, and AMPK activator AICAR protected cells form amiodarone induced cell viability decrease in D407 cells. Artemisinin suppressed the downregulation of p AMPK, Nrf2, and SOD1 protein levels caused by amiodarone, whereas Compound C reversed the effect of artemisinin in D407 cells. The similar results were also obtained in ARPE19 cells and primary human retinal pigment epithelial cells. Collectively, our findings demonstrated that artemisinin could protect human retinal pigment epithelial cells from amiodarone-induced oxidative injury by activating CaMKK2/AMPK/Nrf2 signaling pathway.