VRK1-mediated phosphorylation of histone H3 should be restricted in mitosis for consistent cell cycling, and defects in this process trigger cellular catastrophe. However, an interphasic regulator against VRK1 has not been actually investigated so far. Here, we show that the histone variant macrodomain-containing histone H2A1.2 functions as a suppressor against VRK1 during interphase. The level of macroH2A1.2 was markedly reduced in the mitotic phase, and the macroH2A1.2-mediated inhibition of histone H3 phosphorylation occurred mainly during interphase. We also found direct interaction and binding features between VRK1 and macroH2A1.2 by NMR spectroscopy. Hence, our findings might provide valuable insight into the underlying molecular mechanism regarding an epigenetic regulation of histone H3 during the cell cycle.