Abstract: The aim of this study was to evaluate the role of endogenous nitric oxide (NO) in regulating antioxidant enzyme activity in white clover (Trifolium repens cv. Ladino) under osmotic stress. We investigated endogenous NO fluorescence, dynamic changes in NO content, antioxidant enzyme activity, and the transcript levels of genes encoding antioxidant enzymes in white clover treated with an NO scavenger or NO synthesis inhibitors, and also evaluated the mitigating effects of the exogenous NO donor, sodium nitroprusside (SNP). Leaves of white clover were subjected to osmotic stress (15% PEG-6000). Compared with the leaves in the control, those in the osmotic stress treatment showed higher NO-dependent fluorescence, increased NO content, enhanced activity of antioxidant enzymes, and higher transcript levels of genes encoding antioxidant enzymes. Under osmotic stress, bovine hemoglobin (Hb), sodium vanadate (NaVO₃), and LNG-nitro-L-arginine methyl ester hydrochloride (L-NAME) inhibited the accumulation of NO, leading to inhibition of antioxidant enzyme activity and reduced transcript levels of their encoding genes. The NO donor SNP (50 μmol/L) significantly alleviated the decline in the relative water content (RWC), reduced electrolyte leakage, decreased malondialdehyde accumulation, and promoted antioxidant enzyme activities under osmotic stress. These results suggested that NO is an important signaling molecule in response to osmotic stress, and that it plays a role in regulating gene expression and antioxidant enzyme activity to reduce peroxidation injury in white clover.