Background <p>The role of 2-hydroxyglutarate (2HG) in the methylation process of O6-methylguanine-DNA methyltransferase (MGMT) promoter remains unclear. This study aimed to investigate the predictive efficacy and role of 2HG in MGMT promoter methylation.</p> Methods <p>Patients who met the inclusion criteria were retrospectively included and divided into MGMT promoter methylation group (M) and MGMT promoter unmethylation group (UM). 2HG, Glutamate (Glu), N-acetylaspartate (NAA), Choline (Cho), 2HG/Creatine (Cr), 2HG/Glu and Cho/NAA were calculated. Intergroup differences were estimated using Mann-Whitney U test and Chi-square test. Subsequently, the receiver operating characteristic (ROC) curves were plotted for evaluating diagnostic efficacy of metabolic indices with statistical differences. Then, the causal steps approach was performed for discussing the mediation effect of 2HG.</p> Results <p>75 patients (male: 52, female: 23, age: 46.81 ± 1.52 years) were included (48 in M, 27 in UM). Compared with UM, M exhibited higher 2HG, 2HG/Cr, 2HG/Glu, Cho/NAA and lower Glu, the differences were statistically significant (<i>P</i> &lt; 0.05). 2HG/Glu exhibited the best diagnostic efficacy (area under curve: 0.788, [95%CI: 0.679–0.879]). 2HG is a mediator in the process of MGMT promoter methylation.</p> Conclusions <p>2HG/Glu exhibited the best efficacy in predicting MGMT promoter methylation, and 2HG is a mediator in the methylation process of MGMT promoter.</p>