In this study, to investigate the role of cyclic adenosine diphosphate (ADP)-ribose (cADPR) in nitric oxide–guanosine 3?,5?-cyclic monophosphate (NO–cGMP)-induced isoflavone accumulation in soybean sprouts under ultraviolet B (UVB) radiation, the sprouts were treated with donors and inhibitors of NO and cGMP as well as the cADPR inhibitor. The results showed that NO, with cGMP as a second messenger, activated ADP-ribosyl cyclase, leading to cADPR accumulation under UVB radiation. The cADPR inhibitor suppressed UVB radiation-induced isoflavone synthesis, the activity, gene, and protein expression of chalcone synthase (CHS) and isoflavone synthase (IFS), while this inhibition could be reversed by sodium nitroprusside and 8-Br-cGMP. This suggested that cADPR induced by the NO–cGMP pathway was involved in isoflavone synthesis by elevating the activity, gene, and protein expression of CHS and IFS. Overall, cADPR mediates NO–cGMP-induced isoflavone accumulation in soybean sprouts under UVB stress.