In the present study, in order to analyze abnormalities in DNA methylation in glioma, we utilized a large cohort methylation microarray (119 glioma samples). Genes associated with tumor grade progression were screened through Significance Analysis of Microarrays (SAM) in the methylation microarray. We found that Rab27b was hypomethylated in high-grade glioma (anaplastic gliomas and glioblastomas) compared with low-grade glioma (astrocytoma, oligodendrocytoma and oligoastrocytoma) (p=0.02). In 52 glioma samples, we determined both the methylation status of the Rab27b promoter region and protein expression, and confirmed a negative correlation between the methylation status and expression (p<0.01). Immunohistochemistry of 91 gliomas revealed that the Rab27b expression scores of high-grade glioma were higher than scores of low-grade gliomas (p<0.01). In high-grade gliomas, patients haboring Rab27b hypomethylation or overexpression had unfavorable survival prognosis. Transwell invasion assays identified that invasive cell number of glioma U87 and LN229 cells decreased when Rab27b was knocked down. Decreased invasion partly resulted from reduced expression and activation of MMP-9 after Rab27b knockdown. Downregulation of Rab27b also suppressed tumor growth in vivo. Hypomethylated Rab27b was identified as a progression-associated and prognostic molecular marker of glioma.