Lysine acetylation emerging as a ubiquitous and conserved posttranslational modification plays an important regulatory role in almost every aspect of eukaryotes and prokaryotes. To gain insight into the nature, extent and biological function of lysine acetylation in Beauveria bassiana, a filamentous entomopathogenic fungus, we used immunoaffinity-based acetyl-lysine peptide enrichment integrated with high resolution mass spectrometry to comprehensively characterize lysine acetylated proteins in this fungus. Here we identified a total of 283 proteins with 464 acetylated sites, representing the first acetylproteome reported to date in filamentous fungi. Bioinformatics analysis of this acetylome showed that the acetylated proteins are involved in a wide range of cellular functions, such as metabolism, transcription, and exhibit diverse subcellular localizations. Enrichment of molecular function, biological process, and KEGG pathway implied that identified acetylated proteins of B. bassiana were very important in chromatin organization, ribosome, nucleosome assembly, carbon metabolism, and biosynthesis of secondary metabolites. Moreover, we matched five conserved lysine acetylated motifs containing of KacY, KacH, KacF, FxKac, KacxxxxK and one specific motif KacW in B. bassiana. Taken together, our acetylome analysis revealed a surprising breadth of cellular processes affected by lysine acetylation and also furnishes some fresh intervention nodes for the rational improvement of the friednly entomopathogenic fungus.