This study examined whether sugars and hexose phosphorylation participate in the regulatory mechanisms that induce desiccation tolerance (DT) in seeds. In germinated desiccation‐sensitive radicles of Cucumis sativa and Medicago truncatula, DT was re‐established by an osmotic treatment using polyethylene glycol (PEG) for several days. In cucumber, Glc kinase activity (EC 2.7.1.1) transiently peaked early during PEG incubation before the induction of DT in protruded radicles, whereas Fru kinase activity (EC 2.7.1.4) increased progressively during the re‐establishment of DT. Glucosamine (GAM, a competitive inhibitor of HXK) was able to repress the PEG‐induced DT in both species, whereas hexose and poorly metabolizable hexose analogues had no effect. GAM was effective in repressing DT only early during PEG incubation, indicating that this effect is transient. Both Glc and Man fully rescued GAM‐inhibited DT. PEG‐induced accumulation of Suc was not affected by GAM. Isocitrate lyase (ICL) gene expression, which is known to be regulated by hexoses, responded to the re‐establishment of DT and GAM feeding. In cucumber, expression of ICL was repressed after 6 h of PEG incubation whereas GAM feeding led to ICL de‐repression. When GAM could no longer inhibit the re‐establishment of DT, neither were steady‐state levels of ICL influenced. The implication of HXK as a catalytic regulator and sugar‐sensor in DT is discussed.