Mitogen-activated protein kinase (MAPK) cascades are critical components of signal transduction pathways; however, little is known about whether MAPK is involved in phenylpropanoid pathways under UVB radiation. In this study, we investigated the role of MAPK in mediating nitric oxide (NO) induced isoflavone accumulation in soybean sprouts under UVB radiation. UVB-triggered NO stimulated gene and protein expression of MAPK. Furthermore, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene] (a MAPK inhibitor) hindered UVB-induced isoflavone accumulation and gene and protein expression of enzymes involved in isoflavone biosynthesis [chalcone synthase (CHS) and isoflavone synthase (IFS)]. The attenuation was reversed by exogenous NO donor sodium nitroprusside (SNP). SNP treatment used alone could also enhance isoflavone synthesis and gene and protein expression of CHS and IFS. Overall, MAPK mediates NO-induced isoflavone accumulation in soybean sprouts under UVB radiation.