<p>Lysine methylation is an important post-translational modification that regulates gene transcription, epigenetics, and cell proliferation. Dysregulation of lysine methylation is often observed in cancer cells. Therefore, it is important to understand how cells regulate lysine methylation by lysine methyltransferases and lysine demethylases. However, there are still some biologically essential lysine methylation sites without known demethylases, such as H4K20me3 and H3K79me3. Jumonji-C domain-containing lysine demethylases (JmjC KDMs), as non-heme iron oxygenases, erase methyl marks by hydroxylation of methyl groups, followed by the release of formaldehyde. Current KDM research focuses on the identification of substrates from specific KDMs, but there is a lack of good methods to identify KDM from specific lysine methylation sites. Here, we report a novel strategy to develop activity-based probes that selectively crosslink JmjC-demethylases, which was validated by crosslinking of KDM4A/4D and KDM5B in this study. Allyllysine-containing peptides, as methyllysine substrate mimics, that were prepared by simple cysteine alkylation, are converted to epoxide by demethylase catalysis. The electrophilic intermediate subsequently crosslinks residues inside the KDM catalytic pocket. Therefore, such probes that selectively conjugate KDMs could be applied to identify KDMs from specific methyllysine sites and uncover new biology of lysine methylation.</p>