Phosphorylation of Tyr705 and Ser727 of signal transducer and activator of transcription 3 (STAT3) are known to be required for maximal activation by diverse stimuli. Tyr705 phosphorylation is generally accepted to be mediated by the Janus kinase family. But the mechanism for STAT3 (Ser727) phosphorylation is not well understood. Here, we provide evidence that UVA-induced phosphorylation of STAT3 at Ser727 is inhibited by pretreatment of JB6 cells with PD98059 or SB202190. Phosphorylation of STAT3 (Ser727) is also markedly prevented by a dominant negative mutant of ERK2, c-Jun N-terminal kinase 1 (JNK1), or p38 kinase and in knockout Jnk1-/- or Jnk2-/- cells. Furthermore, STAT3 (Ser727) phosphorylation is suppressed by C- or N-terminal "kinase-dead" mutants of mitogen- and stress-activated protein kinase 1 (MSK1), a downstream kinase of ERKs and p38 kinase, and H89, a potential MSK1 inhibitor. In vitro experiments showed that active MSK1 and JNKs, but not ERKs or p38 kinase, phosphorylate STAT3 (Ser727). Additionally, the role of MAPKs in mediating UVA-stimulated DNA binding activity of STAT3 was investigated. Overall, these results suggest that UVA-induced Ser727 phosphorylation of STAT3 may occur through MSK1 and JNKs.