Abstract：[Objective] To investigate the differential anti-leaf spot efficacy of various concentrations of exogenous methyl jasmonate (MeJA) in barley and elucidate its underlying molecular mechanism, thereby providing a theoretical foundation for the application of MeJA in controlling barley leaf spot.Barley spot blotch is a serious pathogen that significantly affects barley yield and quality. [Method]In this study, we used Mengpimai3 (MP3) seedlings as materials and conducted three different treatments:. Treatment A involved inoculating the barley leaves with sterile water without any Bipolaris sorokiniana (the causal agent of the disease),. Treatment B included inoculating the barley leaves with sterile water after treatment,. Treatment C involved treating the barley leaves with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 mmol/L) of methyl jasmonate (MeJA) before inoculation with the pathogen. The disease incidence was investigated at the three-leaf stage based on disease index and induced resistance effect to determine the optimal concentration of MeJA .Subsequently, we measured antioxidant enzyme activity, anti-disease related enzyme activity, malondialdehyde, proline, soluble sugar, soluble protein, the expression levels of related genes inwith MP3 leaves under no inoculating treatment, inoculation treatment and inoculating + optimal concentration of MeJA treatment were measured at different time points. treated with A, B, and the optimal concentration MeJA of C at different infection times to preliminarily explore differences in MeJA-induced resistance against spot blotch in barley and its molecular mechanism .[Results] The results showed that: (1) Exogenous application of MeJA enhanced MP3''s resistance against spot blotch; particularly at a concentration of 1.5 mmol/L where there was a significant reduction (P<0.05) in disease index by 19.03% compared to control plants, indicating the most effective induction of resistance.. (2) 2) In comparison to inoculating treatment alone, barley leaves treated with 1.5 mmol/L MeJA exhibited significantly increased activities of superoxide, peroxidase, catalase, chitinase and β-,1,3-glucanas. Additionally, there was a significant decrease in malondialdehyde content as well as proline, soluble sugar and soluble protein levels. Furthermore, the expression of MeJA regulated transcription factors and genes encoding disease-resistance related enzymes were significantly upregulated.Treatment C at a concentration of 1.5 mmol/L significantly increased superoxide dismutase, peroxidase, catalase, chitinase and β-1,3-glucanase activity compared to treatment B. The contents of malondialdehyde, proline, soluble sugars, and soluble proteins were significantly decreased (P<0.05); The gene expression levels of MeJA regulated transcription factors and encoding disease-resistant enzymes significantly upregulated (P<0.05). [Conclusion]Therefore, eExogenous application of 1.5 mmol/L MeJA enhances resistance against spot blotch in barley plants by regulating the activity of disease-related enzymes and the content of osmotic regulatory substances, as well as modulating the expression of disease-resistant enzyme genes and key transcription factor genes involved in jasmonic acid signaling pathway.