Abstract: As a newly discovered posttranslational modification (PTM), crotonylation has been identified in histone and nonhistone proteins and has important roles in the regulation of various diseases and biological processes. With the development of high-resolution mass spectrometry approaches, an increasing number of endogenous crotonylation has been detected and identified. The discovery of crotonylation regulatory enzymes paved the way for the study of crotonylation mechanism. In this review, we summarize the biological functions and regulatory mechanism of crotonylation in eukaryotes and introduce the recent progress on crotonylation in malignant tumor.