Dioscorea bulbifera L. is a traditional Chinese herb. As its principal active component, diosbulbin B (DSB) exhibits anticancer activity by inducing cell cycle arrest and apoptosis. Cell cycle progression relies on chromatin dynamics, which is regulated by histone post-translational modifications (PTMs). DSB is metabolized by CYP3A, resulting in the formation of a cis-enedial reactive intermediate (DDE) that selectively and covalently modifies lysine residues on histones, forming pyrroline derivatives, or in cooperation with glutathione (GSH), forming pyrrole derivatives. These modifications competitively disrupted canonical histone PTMs, accompanied by chromatin decompaction. The formation of these histone adductions and the alteration of histone PTM landscapes were validated in both mouse primary hepatocytes and Cyp3a4-HepG2 cells. Molecular dynamics simulations revealed that DDE-derived lysine adductions impair histone-DNA interactions. The observed covalent modification of histone lysine residues provides a novel strategy by regulating chromatin architecture and a conceptual framework for developing antitumor agents based on epigenetic mechanisms.