Growing number of long noncoding RNAs (lncRNAs) are emerging as new modulators in cancer origination and progression. A lncRNA, ADAM metallopeptidase with thrombospondin type 1 motif, 9 (ADAMTS9) antisense RNA 2 (ADAMTS9-AS2), with unknown function, is the antisense transcript of tumor suppressor ADAMTS9. In the present study, we investigated the expression pattern and functional role of ADAMTS9-AS2 in glioma by using real-time PCR and gain-/loss-of-function studies. The results showed that the ADAMTS9-AS2 expression was significantly downregulated in tumor tissues compared with normal tissues and reversely associated with tumor grade and prognosis. Multivariate analysis of the prognosis factors showed that low ADAMTS9-AS2 expression was a significant independent predictor of poor survival in glioma. Overexpression of ADAMTS9-AS2 resulted in significant inhibition of cell migration in glioma, whereas knockdown of ADAMTS9-AS2 showed the opposite effect. We also found that ADAMTS9-AS2 expression was negatively correlated with DNA methyltransferase-1 (DNMT1). In addition, DNMT1 knockdown led to remarkable enhancement of ADAMTS9-AS2 expression. By 5-aza-dC treatment, the ADAMTS9-AS2 expression was also reactivated. The results suggested that ADAMTS9-AS2 is a novel tumor suppressor modulated by DNMT1 in glioma. LncRNA ADAMTS9-AS2 may serve as a potential biomarker and therapeutic target for glioma.