We report the identification of a new type of histone mark, lysine 2-hydroxyisobutyrylation (K hib), and identify the mark at 63 human and mouse histone K hib sites, including 27 unique lysine sites that are not known to be modified by lysine acetylation (K ac) and lysine crotonylation (K cr). This histone mark was initially identified by MS and then validated by chemical and biochemical methods. Histone K hib shows distinct genomic distributions from histone K ac or histone K cr during male germ cell differentiation. Using chromatin immunoprecipitation sequencing, gene expression analysis and immunodetection, we show that in male germ cells, H4K8 hib is associated with active gene transcription in meiotic and post-meiotic cells. In addition, H4K8 ac -associated genes are included in and constitute only a subfraction of H4K8 hib -labeled genes. The histone K hib mark is conserved and widely distributed, has high stoichiometry and induces a large structural change. These findings suggest its critical role on the regulation of chromatin functions.