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Vaccinia related kinases (VRKs) are a class of mammalian Ser/Thr kinase enzymes those have similarity to vaccinia virus B1R kinase. Recently we have characterized that VRK1 phosphorylates histone H3 both at Thr3/Ser10 during mitosis. Here we show that MAP kinase phosphatase 2 (MKP2) binds to VRK1 and suppresses kinase activity of VRK1 in chromatin. We searched for the VRK1 binding partner using GST pull-down assay and found MKP2 as one of candidates. Direct interaction between VRK1 and MKP2 was confirmed with co-immunoprecipitation. Importantly, MKP2 strongly suppressed VRK1 kinase activity in the phosphorylation of core histones dependent on the cell cycle progression. Taken together our results suggest that MKP2 phosphatase activity is required for the proper cell cycle transition, especially during mitosis.