Lysine crotonylation is a newly discovered post‐translational modification, which is structurally and functionally different from the widely studied lysine acetylation. Recent advances in the identification and quantification of lysine crotonylation by mass spectrometry have revealed that non‐histone proteins are frequently crotonylated, implicating it in many biological processes through the regulation of chromatin remodelling, metabolism, cell cycle and cellular organization. In this review, we summarize the writers, erasers and readers of lysine crotonylation, and their physiological functions, including gene transcription, acute kidney injury, spermatogenesis, depression, telomere maintenance, HIV latency and cancer process. These findings not only point to the new functions for lysine crotonylation, but also highlight the mechanisms by which crotonylation regulates various cellular processes.