MAPK (mitogen-activated protein kinase) family plays diverse roles in plant signal transduction and response to abiotic stress. In this study, we isolated a novel MAPKKK gene, GhMAPKKK49 from cotton (Gossypium hirsutum L.). The full-length cDNA of GhMAPKKK49 is 1,056 bp, encoding a protein of 351 amino acids. This protein contains a kinase catalytic domain, composed of an ATP binding site, a Ser/Thr kinase active site and a conserved Raf motif. The alignment analysis revealed a high sequence similarity of GhMAPKKK49 and other plant MAPKKK proteins. Our results indicated a constitutive expression of GhMAPKKK49 in roots, stems, and leaves. The expression of GhMAPKKK49 transcripts was found to be enhanced by abiotic salt, drought and wounding stresses but not the biotic stress of Ralstonia solanacearum or Rhizoctonia solani infection. Furthermore, real-time PCR showed that GhMAPKKK49 is upregulated in response to the application of various signaling molecules, including abscisic acid (ABA), hydrogen peroxide (H2O2), gibberellins (GAs), methyl jasmonate (MeJA), salicylic acid (SA), 6-benzylaminopurine (6-BA), a-naphthylacetic acid (NAA) and ethylene (ET). Furthermore, we demonstrate that GhMAPKKK49 interacts with GhMAPKK4 and GhMAPKK9. Give all that, GhMAPKKK49 may responds to various stresses and may involved in the ABA-, JA-, ET-, SA- and H2O2-mediated signaling pathways.