The essence of human body’s complexity and cellular diversity lies in the plasticity of its genome, shared by all various cell types. Such tremendous plasticity is largely attributed to the alternative deposition of histone variants and epigenetic marks on histone tails, known as post translational modifications (PTMs). We explored the PTMs and corresponding epigenetic “readers” and “writers” of a tissue-specific histone variant H2A.B, whose function and modulatory mechanism were poorly understood despite the pronounced progress in the past two decades. Our proteomic inspection of PTMs on the ectopically expressed H2A.B in Hodgkin’s lymphoma cells by LC-MS/MS mapped several modification sites for post-translational methylation and phosphorylation. Based on this critical information, a peptide pull-down assay incorporating the stable isotope labelling with amino acids in cell culture (SILAC) and LC-MS/MS was performed, resulting in unveiling the interactome of non-modified and modified H2A.B. Among these, we elucidated PTM writers as well as a chromatin remodelling complex and histone chaperon thus, for the first time, identifying the mechanism of H2A.B deposition into the chromatin, as well as establishing that H2A.B has various functions, which are undertaken by specialized PTM variants. Our finding sheds light on the biological roles and modulatory mechanism of histone variant H2A.B in a context of cancer.