The purpose of our study is to clarify the effects of microRNA-129-5p (miR-129-5p) in cellular processes correlated with cancer development and progression of Glioblastoma (GBM) cell by regulating FNDC3B. MiR-129-5p and FNDC3B expression in GBM tissues and tumor adjacent tissues were tested by quantitative real-time PCR. We validated the target relationship between miR-129-5p and FNDC3B by dual luciferase reporter gene system. MTT, colony formation, flow cytometry, Transwell and wound healing assays were used to analyze cell viability, proliferation, apoptosis, invasiveness and migration. The level of FNDC3B protein expression was detected by Western Blot. MiR-129-5p was downregulated in GBM tissues and cell lines, while FNDC3B was upregulated in GBM tissues. The result of luciferase reporter gene assay demonstrated that miR-129-5p could target FNDC3B by binding to the 3′ UTR. The overexpression of miR-129-5p or the inhibition of FNDC3B can both inhibit U87 cell viability, proliferation, migration and invasion, while promote cell apoptosis. Our results suggested that miR-129-5p could directly suppress FNDC3B, which might be one of potential mechanisms in inhibiting cell processes including viability, proliferation, migration and invasiveness of U87 cells.