The regulation effect of melatonin on water use efficiency of leaf and potential mechanisms related to phytohormone of leaf/root were investigated using two wheat (Triticum aestivum L.) cultivars with contrasting drought tolerance: drought-tolerant 'Hengguan35' (HG35) and growing in irrigated fields 'Jimai22' (JM22). Four treatments, including normal water treatment (N), 20% polyethylene glycol (PEG) (P), P + 1 μM melatonin and P + 10 μM melatonin were conducted. Results indicated that exogenous melatonin could significantly improve net photosynthetic rate (Pn), instantaneous water use efficiency (WUE inst. ) and intrinsic water use efficiency (WUE intr. ) of cultivar 'Jimai22'. This was possibly related to increasing root auxin (IAA), zeatin riboside (ZR) content and inhibiting abscisic acid (ABA), hydrogen peroxide (H 2 O 2 ) and aminocyclopropane-1-carboxylic acid (ACC) production in leaf. However, for cultivar 'Hengguan35', 1 μM and 10 μM melatonin did not improve photosynthetic rate and chlorophyll content even took negative effect on that. This could be caused by high vapour pressure deficit (VPD) and high H 2 O 2 contents in leaf. Also, as a drought-tolerant cultivar, 'Hengguan35' possesses some physiological regulation itself such as increasing IAA and ZR content in root and leaf, inhibiting H 2 O 2 production in root. But ABA and ACC content in root was not inhibited, even increased with melatonin under drought treatment. ACC was a potential central player in the hormone cross-talks that regulated leaf and root growth and physiological function. These results suggest that the effect of exogenous melatonin application on drought-resistance in seedling was distinct owing to wheat cultivars with different drought sensitivity, and this was also involved in complicated mechanism of physiological regulation to keep water status. Suitable application of melatonin thus can be an effective way in improving plant drought tolerance in wheat.