The aberrant expression of microRNA-375 (miR-375) has been proved to be associated with carcinogenesis. However, the role of miR-375 in glioblastoma (GBM) remains unknown. The aim of this study was to investigate biological functions and its molecular mechanisms of miR-375 in GBM cells. In this study, real-time PCR results showed that the level of miR-375 expression in GBM tissues and GBM cell lines (U87 and U251) was decreased. Using MTT assay, Transwell migration and invasion assay, we demonstrated that miR-375 overexpression significantly suppress cell proliferation, cell migration and cell invasion capacity in U87 and U251 cells. However, downregulation of miR-375 had reverse effects on cell proliferation, migration and invasion. Targeting association analysis, dual luciferase assay, RT-PCR and western blot analysis results confirmed that miR-375 could target the 3'UTR of Wnt5a mRNA and regulated its protein expression. Further studies also find overexpression of Wnt5a could significantly reverse miR-375-mediated proliferation, migration and invasion on U87 and U251 cells. Therefore, we concluded that miR-375 inhibited the proliferation and invasion of GBM by regulating Wnt5a and might be a possible therapeutic agent for GBM.