Abstract The possible involvement of polyamines (PAs) in the chilling tolerance of cucumber (Cucumis sativus L. cv Jinchun No. 3 and cv Suyo) was investigated. Plants with the first expanded leaves were exposed to 3°C or 15°C in the dark for 24 h (chilling), and then transferred to 28°C/22°C under a 12-h photoperiod for another 24 h (rewarming). Chilling-tolerant cv Jinchun No. 3 showed a marked increase of free spermidine (Spd) in leaves, once during chilling and again during rewarming. Putrescine increased significantly during rewarming, but the increase of spermine was slight. Any of these PAs did not increase in chilling-sensitive cv Suyo during either period. PA-biosynthetic enzyme activities appear to mediate these differences between cultivars. Pretreatment of Spd to cv Suyo prevented chill-induced increases in the contents of hydrogen peroxide in leaves and activities of NADPH oxidases and NADPH-dependent superoxide generation in microsomes and alleviated chilling injury. Pretreatment of methylglyoxal-bis-(guanylhydrazone), a PA biosynthesis inhibitor, to chilled cv Jinchun No. 3 prevented Spd increase and enhanced microsomal NADPH oxidase activity and chilling injury. The results suggest that Spd plays important roles in chilling tolerance of cucumber, probably through prevention of chill-induced activation of NADPH oxidases in microsomes.