Stimulation of p21‐activated kinase‐1 (Pak1) signaling promotes motility, invasiveness, anchorage‐independent growth and abnormal mitotic assembly in human breast cancer cells. Here, we provide new evidence that, before the onset of mitosis, activated Pak1 is specifically localized with the chromosomes during prophase and on the centrosomes in metaphase and moves to the contraction ring during cytokinesis. To identify mitosis‐specific substrates of Pak1, we screened a synchronized G2–M expression library by using a glutathione transferase Pak1 solid‐phase‐based kinase reaction. This analysis identified histone H3 as a substrate of Pak1 both in vitro and in vivo, and it specifically interacted with Pak1 but not Pak2 or Pak3. Site‐directed mutagenesis indicated that Pak1 phosphorylates histone H3 on Ser10. Expressions of the wild‐type, or catalytically active, Pak1 caused it to appear at the poles corresponding to mitotic centrosomes in a variety of mammalian cells. Together, these results suggest for the first time that Pak1 interacts with and phosphorylates histone H3 and may thus influence the Pak1–histone H3 pathway, which in turn may influence mitotic events in breast cancer cells.