Hydrogen peroxide (H2O2) functions as a signal molecule in plants under abiotic and biotic stresses. Leaves of detached maize (Zea mays L.) seedlings were used to study the function of H2O2 pretreatment in osmotic stress resistance. Low H2O2 concentration (10 mM) which did not cause a visual symptom of water deficit (leaf rolling) was applied to the seedlings. Exogenous H2O2 alone increased leaf water potential, endogenous H2O2 content, abscisic acid (ABA) concentration, and metabolite levels including soluble sugars, proline, and polyamines while it decreased lipid peroxidation and stomatal conductance. Osmotic stress induced by polyethylene glycol (PEG 6000) decreased leaf water potential and stomatal conductance but enhanced lipid peroxidation, endogenous H2O2 content, the metabolite levels, and ABA content. H2O2 pretreatment also induced the metabolite accumulation and improved water status, stomatal conductance, lipid peroxidation, ABA, and H2O2 levels under osmotic stress. These results indicated that H2O2 pretreatment may alleviate water loss and induce osmotic stress resistance by increasing the levels of soluble sugars, proline, and polyamines thus ABA and H2O2 production slightly decrease in maize seedlings under osmotic stress.