Acetylation has been demonstrated to be a global post-translational modification. However, identification of acetylation sites in Arabidopsis is insufficient and its ethylene-induced changes in acetylome remains unknown. Here we use a robust quantitative proteomics strategy which is dimethyl-label based extracted ion chromatogram quantitation to monitor the acetylome changes after long-term ethylene treatment in Arabidopsis EIN3 (Ethylene Insensitive 3)/EIL1 (Ethylene Insensitive 3-Like 1) double mutants. In total, 6,431 acetylation sites were identified from 3,039 proteins. Among them, 30 ethylene-enhanced and 27 ethylene-suppressed acetyl-peptides were quantified from various proteins, including histone proteins, ribosomal proteins and heat shock proteins. Long-term ethylene treatment led to smaller rosette diameters and shorter root lengths in both wild type and ein3/eil1 mutants of Arabidopsis, which indicates that ethylene can regulate plant growth and development by both EIN3/EIL1-dependent and –independent ways. According to the quantitation results, these regulation may happen during the processes of gene transcription, protein translation and protein folding.