MicroRNAs (miRNAs/miRs) serve tumor promoting or suppressive roles in different human cancer types, including glioma; however, the regulatory underlying mechanism by which miR-599 affects glioma progression remains largely unknown. The aim of the present study was to investigate the expression of miR-599 in glioma, as well as the underlying regulatory mechanism. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to examine mRNA and protein expression, respectively. MTT, wound healing and transwell assays were conducted to study cell proliferation, migration and invasion, respectively. A dual-luciferase reporter gene assay was used to confirm the targeting association between miR-599 and Ras-related protein Rab-27B (hereafter RAB27B). In the present study, miR-599 expression was observed to be significantly downregulated in human glioma tissues and cell lines, when compared with normal brain tissues and normal human astrocyte cells, respectively. Low miR-599 expression was significantly associated with glioma progression. Ectopic expression of miR-599 caused a significant reduction in the proliferation, migration and invasion of U-87MG Uppsala and U251 cells. Bioinformatics analysis and dual-luciferase reporter gene assay data identified that RAB27B was a direct target gene of miR-599. The mRNA and protein expression of RAB27B was significantly downregulated following miR-599 overexpression in U-87MG Uppsala and U251 cells. Rescue experiments demonstrated that RAB27B rescued the miR-599-induced inhibition of glioma cell growth, migration and invasion. In addition, RAB27B expression was significantly upregulated in glioma tissues and cell lines, and the expression levels of RAB27B were inversely correlated with miR-599 levels in glioma tissues; therefore, the present study demonstrated that miR-599 exerts a tumor-suppressive role in glioma progression by targeting RAB27B, indicating that miR-599 may be used as a potential candidate for glioma treatment.