Drought stress is a major challenge in agriculture, causing severe loss of crops throughout the world. Methyl jasmonate (MeJA) regulates a variety of plant developmental process and responds to many biotic and abiotic stresses. To explore the physiological mechanisms of drought stress mitigated by exogenous MeJA, we subjected callus of wheat (Triticum aestivum L.) to drought induced medium after pretreatment with 10 ml sterile solution that contained the following singly or in combinations: MeJA (0, 0.25, and 2.5µM) and the lipoxygenase (LOX) inhibitor salicylhydroxamic acid (SHAM; 0 and 0.5 mM) for 24 hours. The endogenous jasmonic acid (JA) content, activity of LOX, hydrogen peroxide (H2O2) content, antioxidant enzymes activities, levels of reactive oxygen species (ROS), content of malondialdehyde (MDA), and cell viability were measured. The results showed that exogenous MeJA induced the activity of LOX and resulted in rapid increase in endogenous JA levels in a concentration-dependent manner; JA declined gradually after reaching its maximum. The level of H2O2 increased with increases in LOX activity and endogenous JA level, which are involved in the octadecanoid signaling pathway. However, SHAM inhibited the LOX activity, the endogenous JA level, and the accumulation of H2O2 in wheat callus. In addition, the activities of several antioxidant enzymes increased after MeJA-pretreatment, but this effect was also inhibited by SHAM. The cumulative results suggest that H2O2 was generated through the octadecanoid signaling pathway, and some antioxidant defense genes were provoked by the accumulation of H2O2. Therefore, we inferred that H2O2 might act as a signaling molecule for the MeJA-induced antioxidant defense. Drought stress can be improved by MeJA-pretreatment, leading to decrease in endogenous MDA and ROS contents, which are normally induced by drought conditions. Both drought stress and exogenous MeJA-pretreatment increased in LOX activity, endogenous JA level, and antioxidant enzymes. Combinatorial treatments showed an apparent synergistic effect on the activities of these antioxidant enzymes and resulted in improved cell viability. These results confirmed the hypothesis that H2O2 acts as a signaling molecule for the MeJA-induced antioxidant defense and could alleviate the negative effects of drought stress on wheat callus.