Ionic liquids (ILs) are extensively used in various fields, posing a potential threat in the ecosystem because of their high stability, excellent solubility, and biological toxicity. In this study, the toxicity mechanism of three ILs, 1-octyl-3-methylimidazolium chloride ([CMIM]Cl), 1-decyl-3-methylimidazolium chloride ([CMIM]Cl), and 1-dodecyl-3-methylimidazolium chloride ([CMIM]Cl) on Arabidopsis thaliana were revealed. Reactive oxygen species (ROS) level increased with higher concentration and longer carbon chain length of ILs, which led to the increase of malondialdehyde (MDA) content and antioxidase activity, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and peroxidase (POD) activities. SOD, CAT, and GPX activities decreased in high ILs concentration due to the excessive ROS. Differentially expressed protein was analyzed based on Gene ontology (GO) and KEGG pathways analysis. 70, 45, 84 up-regulated proteins, and 72, 104, 79 down-regulated proteins were identified in [CMIM]Cl, [CMIM]Cl, and [CMIM]Cl treatment, respectively (fold change ≥ 1.5 with ≥95% confidence). Cellular aldehyde metabolic process, mitochondrial and mitochondrial respiratory chains, glutathione transferase and oxidoreductase activity were enriched as up-regulated proteins as the defense mechanism of A. thaliana to resist external stresses. Chloroplast, photosynthetic membrane and thylakoid, structural constituent of ribosome, and transmembrane transport were enriched as the down-regulated protein. Compared with the control, 8 and 14 KEGG pathways were identified forup-regulated and down-regulated proteins, respectively, in three IL treatments. Metabolic pathways, carbon metabolism, biosynthesis of amino acids, porphyrin and chlorophyll metabolism were significantly down-regulated. The GO terms annotation demonstrated the oxidative stress response and effects on photosynthesis of A. thaliana in ILs treatment from biological process, cellular component, and molecular function categories.