Abstract: A pharmacological experiment was designed to investigate the function of nitric oxide (NO) in the spermidine-induced antioxidant enzyme activities and gene expression in white clover (Ladino). Treatment with exogenous spermidine (Spd) at 20 μmol/L significantly improved (P<0.05) the nitrate reductase (NR) and nitric oxide synthase (NOS) activities in leaves of white clover and NO content reached a maximum after 2 h of Spd treatment. However, treatments that included 50 μmol/L bovine hemoglobin (Hb) (NO-scavenger), 5 mmol/L partial sodium vanadate (NaVO₃) (a NR-inhibitor) and 200 μmol/L NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a NOS-inhibitor) inhibited the accumulation of NO in Spd-treated white clover. Exogenous Spd treatment can also enhance the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbic acid peroxidase (APX) and the relevant gene expression in leaves. Hb, NaVO₃ and L-NAME inhibited the Spd-induced enzyme activities of SOD, POD, CAT, APX and gene expression to different degrees. These results suggest that Spd induced NO by enhancing antioxidant enzyme activities and expression of related genes in white clover, and that NO production is probably regulated from the NR and NOS pathways.