Lysine crotonylation on histones is a recently identified post-translational modification that has been demonstrated to associate with active promoters and to directly stimulate transcription.Given that crotonyl-CoA is essential for the acyl transfer reaction and it is a metabolic intermediate widely localized within the cell, we postulate that lysine crotonylation on non-histone proteins could also widely exist. Using specific antibody to enrich crotonylated lysine(Kcr) peptides followed by high-resolution mass spectrometry analysis reveals that crotonylated proteins and lysine residues. Bioinformatic analysis reveals that crotonylated proteins are particularly enriched in the nuclei, that lysine crotonylation alters level of the modified proteins in the chromatin and that cotonylation of a subset of proteins influences DNA replication and cell cycle. Taken together, our data indicate that lysine crotonylation could be induced in a large number of proteins other than histones and this type of acyl modification could play an important role regulating multiple cellular processes.