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As a suppressor of Hedgehog signaling pathway, microRNA-326 (miR-326) has been demonstrated to control the development of cerebellar neuronal progenitor and tumor cells. More recently, it has been reported that miR-326 was down-regulated in glioblastoma tissues and might regulate the metabolic activity of glioma and glioma stem cells, suggesting the involvement of miR-326 in tumorigenesis and progression of gliomas. However, the role of miR-326 in human glioma has not been clearly understood. Therefore, the aim of this study was to investigate the clinical significance of miR-326 expression in human glioma. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to characterize the expression patterns of miR-326 in 108 glioma and 20 normal brain tissues. The associations of miR-326 expression with clinicopathological factors and prognosis of glioma patients were also statistically analyzed. The expression levels of miR-326 in glioma tissues were significantly lower than those in normal brain tissues (P < 0.001). Additionally, the decreased miR-326 expression in glioma was significantly associated with advanced pathological grade (P = 0.01) and low Karnofsky performance score (KPS, P = 0.03). Moreover, Kaplan-Meier survival and Cox regression analyses showed that low expression of miR-326 (P = 0.01) and advanced pathological grade (P = 0.02) were independent factors predicting poor prognosis for gliomas. Furthermore, subgroup analyses showed that miR-326 expression was significantly associated with poor overall survival in glioma patients with high pathological grades (for grade III-IV: P < 0.001). Down-regulation of miR-326 may have potential value for predicting clinical outcomes in glioma patients with high pathological grades, suggesting that miR-326 is an important candidate tumor suppressor, and its down-regulated expression may contribute to glioma progression.