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In order to develop tolerance-enhanced alfalfa, the hypocotyls of alfalfa (Medicago sativa L.Xinjiang Daye) were used as explants for transformation by Agrobacterium-mediated processes.The expression vector of pCAMB/A2300 contains Arabidopsis nucleoside diphosphate kinase 2 gene AtNDPK2 under the control of an oxidative stress-inducible SWPA2 promoter.Twelve transgenic alfalfa lines were identified by PCR amplification analysis with AtNDPK2 specific primer.RT-PCR results confirm the induced expression of AtNDPK2 by methyl viologen (MV)-mediated oxidative stress.Under 10 μmol/L MV treatment, the relative ion leakage of six transgenic alfalfa lines decreased 32.76%~50.06% and 20.01%~30.96% compared with control plants at 48 h and 72 h respectively.Meanwhile, the oxidative stress-tolerance of transgenic alfalfa plants was found positively associated with the activity of transcripted AtNDPK2 mRNA.We expect that the transgenic alfalfa with enhanced stress tolerance might be useful for sustainable agriculture in marginal soils, including desertified areas and alkalinized soils.