Wnt5a operates as either a tumor suppressor or a tumor stimulator, according to tumor type. The functions of Wnt5a in human glioblastoma (GBM) have yet to be determined. We initially evaluated the expression of Wnt5a in human glioma. The results of immunohistochemical analyses have revealed that Wnt5a expression was higher in human GBM than in normal brain tissue and low-grade astrocytoma. In order to assess the role of Wnt5a on proliferation in human glioblastoma cells, we employed U87MG and GBM-05, a newly established GBM cell line. GBM-05 was established from a patient diagnosed with GBM. GBM-05 cells were shown to express Nestin, but did not express GFAP and Map2ab. GBM-05 cells formed infiltrating brain tumors after being intracerebrally transplanted into nude mice, and xenotransplanted GBM-05 cells were observed to differentiate into neuronal and astrocyte lineages. Wnt5a expression in the xenotransplanted tumors was higher than that detected in the surrounding brain tissues. The overexpression of Wnt5a increased the proliferation of GBM-05 and U87MG in vitro. By way of contrast, the downregulation of Wnt5a expression as the result of RNA interference reduced proliferation from GBM-05 and U87MG cells in vitro, and reduced tumorigenicity in vivo. Our data indicate that Wnt5a signaling is an important regulator in the proliferation of human glioma cells.