In the present study, we performed the first global lysine acetylome in V. mimicus through a combination of highly sensitive immune-affinity purification and high-resolution LC−MS/MS, and identified 1097 lysine-acetylated sites on 582 proteins. Of the acetylated proteins, more than half were acetylated on one site. In the acetylome of V. mimicus, six significantly enriched motifs were identified and obtained including KacL, KacR, L(-2)KacL, LKacK, L(-7)EKac, and IEKac. In addition, bioinformatics results indicated that acetylated proteins participate in many different biological functions in V. mimicus, such as purine metabolism, ribosome, pyruvate metabolism, glycolysis / gluconeogenesis, TCA cycle, and so on. Moreover, 13 acetylated proteins were related to the virulence of V. mimicus.