is an opportunistic pathogen and has become a globally clinical concern due to multidrug resistance. In this study, we generated a series of strains (CF2, -3, -4, -5, and -6) with elevated MIC of fluconazole from CF1 by gradually increasing fluconazole concentration. Employing 4D label-free proteomics, we explored the contributions of acetylome alterations to the evolution of fluconazole resistance in . From 18 samples of six strains, we identified 2419 acetylated proteins with 7833 sites, quantified 3032 proteins, and 6310 sites. Compared to CF1, differentially acetylated proteins were significantly linked to cytosolic ribosomes, structural molecule activity, hexose and monosaccharide catabolism, glycolysis/gluconeogenesis, and alcohol dehydrogenase GroES-like domain. CF3 (16-32 μg/mL) and CF4 (64 μg/mL) were suggested as the key resistance inflection. CF3 was associated with a significant decrease in ribosomal protein acetylation, while CF4 witnesses a marked upsurge in acetylated proteins and sites. Upregulated proteins in CF3 were notably involved in oxidative stress, detoxification, osmotic stress, and alcohol. We drew a diagram to show acetylated sites on histones in . To the best of our knowledge, this is the first acetylome study in , providing insights into the biological and pathogenic roles of post-translational modifications (PTMs) in this organism.