Prior exposure of plants to a triggering factor can enhance their tolerance to more severe stressful events. Transcriptome reprogramming of metabolism and hormonal modulation processes in the resurrection plant Boea hygrometrica was observed during drought acclimation. However, the metabolic dynamics and underlying regulatory networks that modulate drought acclimation-induced rapid desiccation tolerance (RDT) remain unexplored. Here, we performed an integrated transcriptome and metabolome analysis to investigate the phytohormone profiles and metabolic landscapes of B. hygrometrica during drought acclimation and dehydration stress. We identified a set of RDT acquisition-associated biomarkers, including trans-zeatin and some disaccharides (lactose, trehalose, sucrose, and isomaltulose). Exogenous application of lactose effectively enhanced the RDT of B. hygrometrica seedlings and improved drought tolerance in Arabidopsis, tobacco, maize, and wheat. In addition, transient overexpression of lactose-associated transcription factors MYB330 and APETALA2 in B. hygrometrica can promote the RDT and transcription of drought acclimation-inducible genes involved in calcium and ABA signalling and autophagy. In summary, our findings demonstrate that drought acclimation-induced lactose accumulation facilitates the establishment of an “acclimated state”, leading to transcriptome reprogramming in response to rapid desiccation. These results will also pave the way for using RDT biomarkers to improve crop drought tolerance in an environmentally sustainable manner.