The mitogen-activated protein kinase cascades elicit modification of chromatin proteins such as histone H3 by phosphorylation concomitant with gene activation. Here, we demonstrate for the first time that the mixed lineage kinase-like mitogen-activated protein triple kinase (MLTK)-α phosphorylates histone H3 at Ser28. MLTK-α but neither a kinase-negative mutant of MLTK-α nor MLTK-β interacted with and phosphorylated histone H3 in vivo and in vitro. When overexpressed in 293T or JB6 Cl41 cells, MLTK-α phosphorylated histone H3 at Ser28 but not at Ser10. The interaction between MLTK-α and histone H3 was enhanced by stimulation with ultraviolet B light (UVB) or epidermal growth factor (EGF), which resulted in the accumulation of MLTK-α in the nucleus. UVB- or EGF-induced phosphorylation of histone H3 at Ser28 was not affected by PD 98059, a MEK inhibitor, or SB 202190, a p38 kinase inhibitor, in MLTK-α-overexpressing JB6 Cl41 cells. Significantly, UVB- or EGF-induced phosphorylation of histone H3 at Ser28 was blocked by small interfering RNA of MLTK-α. The inhibition of histone H3 phosphorylation at Ser28 in the MLTK-α knock-down JB6 Cl41 cells was not due to a defect in mitogen- and stress-activated protein kinase 1 or 90-kDa ribosomal S6 kinase (p90RSK) activity. In summary, these results illustrate that MLTK-α plays a key role in the UVB- and EGF-induced phosphorylation of histone H3 at Ser28, suggesting that MLTK-α might be a new histone H3 kinase at the level of mitogen-activated protein kinase kinase kinases.