The widespread use of artemisinin (ART) and its derivatives has significantly reduced the global burden of malaria; however, malaria still poses a serious threat to global health. Although significant progress has been achieved in elucidating the antimalarial mechanisms of ART, the most crucial target proteins and pathways of ART remain unknown. Knowledge on the exact antimalarial mechanisms of ART is urgently needed, as signs of emerging ART resistance have been observed in some regions of the world. Here, we used a combined strategy involving mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) and transcriptomics profiling to identify a group of putative antimalarial targets of ART. We then conducted a series of validation experiments on five prospective protein targets, demonstrating that ART may function against malaria parasites by interfering with redox homeostasis, lipid metabolism, and protein synthesis processes. Taken together, this study provides fresh perspectives on the antimalarial mechanisms of ART and identifies several crucial proteins involved in parasite survival that can be targeted to combat malaria.