Mitogen-activated protein kinase (MAPK) cascades are important intracellular signaling modules and function as a convergent point for crosstalk during abiotic stress signaling. In this article, we isolated a novel group B MAPKK gene, ZmMKK3, from Zea mays. ZmMKK3 protein might be localized in both the cytoplasm and the nucleus. RNA blot analysis indicated that the ZmMKK3 transcription was up-regulated by abscisic acid (ABA), hydrogen peroxide (H 2 O 2 ) and PEG, and that H 2 O 2 mediated PEG-induced expression of ZmMKK3. Constitutive expression of ZmMKK3 in Nicotiana tabacum reduced H 2 O 2 accumulation under osmotic stress by affecting antioxidant defense systems and alleviated reactive oxygen species-mediated injury under oxidative stress. Transgenic tobacco exhibited attenuated ABA sensitivity by means of an increased germination rate and main root growth. Taken together, these results indicate that ZmMKK3 is a positive regulator of osmotic tolerance and ABA signaling in plants.