Big mitogen-activated protein (MAP) kinase (BMK1), a member of the mammalian MAP kinase family, is activated by growth factors. The activation of BMK1 is required for growth factor-induced cell proliferation and cell cycle progression. We have previously shown that BMK1 regulates c-jun gene expression through direct phosphorylation and activation of transcription factor MEF2C. MEF2C belongs to the myocyte enhancer factor 2 (MEF2) protein family, a four-membered family of transcription factors denoted MEF2A, -2B, -2C, and -2D. Here, we demonstrate that, in addition to MEF2C, BMK1 phosphorylates and activates MEF2A and MEF2D but not MEF2B. The blocking of BMK1 signaling inhibits the epidermal growth factor-dependent activation of these three MEF2 transcription factors. The sites phosphorylated by activated BMK1 were mapped to Ser-355, Thr-312, and Thr-319 of MEF2A and Ser-179 of MEF2D both in vitro and in vivo. Site-directed mutagenesis reveals that the phosphorylation of these sites in MEF2A and MEF2D are necessary for the induction of MEF2A and 2D transactivating activity by either BMK1 or by epidermal growth factor. Taken together, these data demonstrate that, upon growth factor induction, BMK1 directly phosphorylates and activates three members of the MEF2 family of transcription factors thereby inducing MEF2-dependent gene expression.