This study aims to investigate the relationship between Rab27a and the characteristics of glioma cell U251 such as proliferation, apoptosis, and invasion and to provide an experimental basis for future therapy in human glioma. Recombinant plasmid of pcDNA3.1-Rab27a was constructed and transfected into U251 cells with the help of Lipofectamine™2000. The expression of Rab27a was detected by Western blot. Cell viability, cell cycle, cell apoptosis, and cell migration were analyzed, respectively, by (3-(4,5)-dimethylthi-azol-2-yl)-2,5-diphenytetrazolium bromide (MTT) assay, flow cytometry, and Transwell invasion chamber methods. Meanwhile, the effect of Rab27a on secretion of cathepsin D in U251 cells was also examined. With the help of luciferase reporter assay system, the relationship between miR-124 and gene Rab27a expression was explored. Western blot showed that the expression of Rab27a was significantly increased in pcDNA3.1-Rab27a transfection group (p < 0.01) and that was significantly decreased in Rab27a-shRNA transfection group (p < 0.01) compared with control group. MTT assay, flow cytometry, and Transwell invasion chamber experiment indicated that cell viability (p < 0.01), proliferation index (p < 0.05), and invasion ability (p < 0.01) were improved significantly in pcDNA3.1-Rab27a transfection group compared with control group and that cell viability (p < 0.01), proliferation index (p < 0.05), and invasion ability (p < 0.01) were reduced markedly in Rab27a-shRNA transfection group compared with control group. The apoptosis analysis by flow cytometry demonstrated that the ratio of apoptosis in pcDNA3.1-Rab27a transfection group was significantly lower than that in control group (p < 0.05) and the ratio was notably higher in Rab27a-shRNAtransfection group than that in the control group. Cathepsin D activity assay indicated that the release of cathepsin D was enhanced in pcDNA3.1-Rab27a transfection group compared to that in the control group (p < 0.05). Rab27a could increase the glioma cell ability, promote proliferation and invasion, and suppress cell apoptosis. The above-stated effects of Rab27a possibly were exerted by increasing the secretion of cathepsin D and regulated by miR-124. In addition, the inhibition of expression of Rab27a perhaps benefited the therapy for glioma patients.