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 D407 cells (Human retinal pigment epithelial cells) and to assess if artemisinin could protect D407 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, enzyme activity of caspase 3 was analyzed using Caspase 3 Activity Assay Kit, cell apoptosis was detected by measuring mitochondrial membrane potential (MMP) and Flow Cytometry. The phosphorylation level of AMPK protein, and the expression level of CaMMK2, Nrf2, SOD1 and GAPDH proteins were analyzed by Western blot assay. Our results revealed that artemisinin could attenuate amiodarone-induced cell viability decrease, intracellular ROS level increase and mitochondrial membrane potential (△ψm) decrease, as well as suppress amiodarone-induced caspase 3 activity increase. The artemisinin also upregulated the phosphorylation level of AMPK and protein level of CaMMK2, Nrf2, 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. The similar result was also obtained on primary human retinal pigment epithelial (RPE) cells. Collectively, these results suggested that artemisinin could protect D407 cells from amiodarone-induced oxidative injury through the activation of CaMKK2/AMPK/Nrf2 signaling pathway. Acknowledgement: This research was supported by National Natural Science Foundation of China (File No.31771128 and 32070969), The Science and Technology Development Fund, Macau SAR (File No. 0113/2018/A3, 0044/2019/AGJ and 0127/2019/A3), University of Macau (File No. MYRG2018-00134-FHS)