Big Mitogen-Activated Protein Kinase 1 (BMK1/ERK5) is regulated sequentially by a series of upstream MAP Kinase Kinases (MEKs) in a signaling cascade. MEKs activate their downstream MAPK by phosphorylation of threonine and tyrosine in the T-X-Y motif. MEK5 is the upstream BMK1 kinase and exists as naturally-occurring splice variants, MEK5a and MEK5b. The full-length MEK5 (MEK5a) is 89 amino acids longer than MEK5b at the N-terminus but the precise functional difference between the two splice variants is not known. Dual phosphorylation site mutation of MEK5a (Ser311Asp and Thr315Asp; MEK5a(S311D/T315D)) activated BMK1, but the corresponding dual phosphorylation sites-mutant of MEK5b could not induce BMK1 kinase activation or nuclear translocation. Furthermore, MEK5b inhibited EGF-induced BMK1 activation and MEK5a (S311D/T315D)-induced MEF2 transcriptional activity. Both MEK5a and MEK5b individually co-immunoprecipitated with BMK1, but the presence of MEK5b prevented association of MEK5a with BMK1 suggesting a mechanistic basis for the dominant-negative behavior of MEK5b on BMK1 activation. The ratio of MEK5a to MEK5b expression was higher in cancer cell lines, and overexpression of MEK5b inhibited serum-induced DNA synthesis. These data suggest that alternative splicing of MEK5a and MEK5b may play a critical role in BMK1 activation and subsequent cell proliferation.