Glioma is the most common type of primary central nervous system tumor. Ser/Thr protein phosphatase 5 (PP5) has been shown to regulate multiple signaling cascades that suppress growth and facilitate apoptosis in several human cancer cells. However, the role of PP5 in human gliomas remains unclear. Herein, the relationship between PP5 expression and glioma cell growth was investigated, and the therapeutic value of PP5 in glioma was further evaluated. We employed a short hairpin RNA targeting PPP5C gene to knock down PP5 expression in human glioma cell lines U251 and U373. Depletion of PPP5C via RNAi remarkably inhibited glioma cell proliferation and colony formation, and arrested cell cycle in the G0/G1 phase. Moreover, knockdown of PP5 markedly suppressed glioma cell migration, as determined by Transwell assay. Our findings suggest that PPP5C could be essential for glioma cell growth and serve as a promising therapeutic target in human gliomas.