In Homo sapiens, Streptococcus agalactiae is a common colonizer of the rectovaginal tract and a fundamental cause of neonatal and non-pregnant adults infectious diseases. It also causes infectious disease in fish which compromises food safety as well as possesses a zoonotic risk. Lysine crotonylation (Kcr) is a type of histone post-translational modifications discovered in 2011. Kcr dynamics are involved in active gene promoters and potential enhancers in yeast and mammalian. However, lysine crotonylation in S. agalactiae has not yet been studied. In the present study, we conducted the first proteome-wide profiling of Kcr in S. agalactiae and identified 241 Kcr sites on 675 proteins, representing the Kcr event in S. agalactiae. Bioinformatics analysis showed that 164 sequences were matched to a total of six definitively conserved motifs, and many of them were significantly enriched in metabolic processes, cellular process, and single-organism processes. Moreover, we found four crotonylation modified proteins predicted as quorum sensing system and virulence factors, which indicate the important role of PTM on bacterial QS system and virulence. These data represent the first report of a global crotonylation proteome and provide a promising starting point for further functional research of crotonylation in bacterial virulence in S. agalactiae.