Cucumber (Cucumis sativus L.) plants were subjected to hypoxic stress with or without a pretreatment of putrescine (Put) to investigate whether nitrate reduction is involved in the enhancement effects of Put on tolerance to root-zone hypoxia. Both hypoxic stress and exogenous Put application significantly increased the contents of endogenous Put, spermidine and spermine. Plants grown under hypoxic conditions exhibited reductions in plant growth rate, NAD⁺/NADH ratio, ATP concentration, and consequent lowered cell viability in roots. The detrimental effects, however, were significantly alleviated by the addition of Put into the nutrient solution 24 h before the administration of hypoxia. Transcript levels of NR (nitrate reductase) and its cofactor binding domain genes FAD (FAD binding) and CYP51G1 (Heme binding), the activity of nitrate reductase (NR, EC 1.6.6.1) and the nitrate reduction process were each greatly enhanced by Put application, particularly in roots exposed to hypoxia. Lactate dehydrogenase (EC 1.1.1.27) activity was independent of aeration condition and Put application, whereas alcohol dehydrogenase (EC 1.1.1.1) activity was significantly increased after exposure to hypoxia, but did not increase after Put application. Put failed to alleviate the hypoxia injury of root electrolyte leakage when NR was inhibited by tungstate in the nutrient solution. These results suggest that Put enhances tolerance to hypoxia by increasing the transcript levels of NR and its cofactor binding domain genes, thereby stimulating the activities of NR and nitrate reduction to maintain the redox and energy status.