Trehalose (Tre) plays a vital role in response to drought stress in plants but its regulatory mechanism remains unclear. Here, this study explores the mechanism of re-regulated drought tolerance during cucumber adventitious root formation. Our results indicate that 2 mM Tre displays remarkable drought alleviation in the aspect of root number, root length, fresh weight, and dry weight. Under drought stress, Tre could inhibit greatly the MDA, H 2 O 2 , and O 2 − accumulation, enhance obviously the activities of SOD, POD, and CAT enzymes and up-regulate significantly the transcript levels of SOD , POD , and CAT genes. Furthermore, Tre treatment also promotes Tre metabolism during drought stress: significantly increases starch and Tre contents and decreases glucose content, the biosynthesis enzymatic activity of the Tre metabolic pathway including TPS and TPP are enhanced and the activity of degradation enzyme THL is decreased, and corresponding genes TPS1 , TPS2 , TPPA , and TPPB are up-regulated. Tre significantly reversed the decrease caused by PEG in IAA, ethylene, ABA, and BR contents and the increase caused by PEG in GA 3 and KT contents. Collectively, Tre appears to be the effective treatment in counteracting the negative effects of drought stress during adventitious root formation by regulating ROS, Tre metabolisms and plant hormones. • Trehalose enhances drought stress resistance in cucumber during adventitious rooting. • Trehalose-medicated ROS improves drought stress resistance. • Trehalose alleviates cucumber drought stress by regulating trehalose metabolism. [ABSTRACT FROM AUTHOR]