Abstract: The effects of spermidine (Spd) on relative water content, reactive oxygen, membrane lipid preoxidation, antioxidant enzyme activity and proline metabolism were studied in white clover (Trifolium repens) leaves, cultivar ‘Ladino’. The expression patterns of four different antioxidant enzyme genes were analyzed using semi-quantitative RT-PCR under different water stress. Under water stress, relative water content gradually declined whereas reactive oxygen and malondialdehyde (MDA) content increased. Exogenous Spd significantly improved relative water content and effectively reduced the accumulation of reactive oxygen and MDA. Exogenous Spd changed antioxidant activities and gene expression pattern under different water stress regimes and increased expression of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) genes to improve antioxidant enzyme activities, reducing water stress injury. Exogenous Spd accelerated proline catabolism and biosynthesis from the glutamate pathway during water stress although it had no effect on the ornithine pathway of proline biosynthesis. The results suggested that exogenous Spd can effectively improve water stress tolerance of white clover associated with the improving antioxidant enzyme activities, enhancing expression of genes encoding antioxidant enzymes and accelerating proline metabolism.