Crotonylation (Cr) is a novel post-transcriptional modification with the potential to regulate multiple biological processes. In order to improve the crotonylation dataset for further study, we performed LC-MS/MS with mouse liver samples to obtain the in vivo crotonylome. Totally, we identified 4607 unique crotonylated peptides in 1322 proteins. The motifs KcrE, KcrD and EKcr were significantly enriched with the crotonylated peptides whose localization probability was over 0.75. The identified crotonylated proteins were mostly located in cytoplasm and nucleus, and most crotonylated proteins were enzymes. Functional enrichment analysis based on GO and KEGG pathway showed that, crotonylated proteins were closely related to small molecule catabolic process, purine-containing compound metabolic process, carbon metabolism pathway, ribosome pathway and a series of metabolism associated biological processes. In conclusion, this research provided the largest in vivo dataset of crotonylome and the first crotonylome of mouse liver. We offered another evidence that crotonylation widely existed in non-histone proteins and was probably involved in various biological processes.