Amiodarone is a highly effective anti-arrhythmic drug, but the clinical application of amiodarone is limited by serious adverse effects including ocular toxicity. The purpose of this study was to evaluate the toxic effect of amiodarone on human retinal pigment epithelial cells and to assess if artemisinin could protect cells from amiodarone-induced oxidative damage and apoptosis, and also explore the underlying molecular mechanisms. The cell viability was measured by MTT assay, intracellular reactive oxygen species (ROS) level was assessed using fluorophotometric quantification, cell apoptosis was detected by measuring mitochondrial membrane potential (MMP) or Flow Cytometry. The phosphorylation level of AMPK protein, and the expression level of CaMMK2, Nrf2, SOD1 and GAPDH proteins were analyzed by Western blot analysis. Our results revealed that artemisinin could attenuate amiodarone-induced cell viability decrease, intracellular ROS level increase and mitochondrial membrane potential (△ψm) depolarization in human retinal pigment epithelial cell line D407 cells. The artemisinin also upregulated the phosphorylation level of AMPK as well as protein level of CaMMK2, Nrf2 and SOD1 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 inhibited the artemisinin-induced upregulation of p-AMPK, Nrf2, and SOD1 protein level in D407 cells. Furthermore, the AMPK activator AICAR protected D407 cell from amiodarone-induced cell viability decrease. The similar result was also obtained in human retinal pigment epithelial cell line ARPE19 cells and primary human retinal pigment epithelial cells. Collectively, these findings suggested that artemisinin could protect human retinal pigment epithelial cells from amiodarone-induced oxidative injury through the activation of CaMKK2/AMPK/Nrf2 signaling pathway.