Rapid increases in the tyrosine phosphorylation of signal transducers and activators of transcription 5 (STAT5) proteins have been extensively documented in cells stimulated with cytokines and growth factors. However, the mechanisms by which STAT5 translocates to the nucleus and regulates proliferation in human glioblastoma multiforme cells have not been studied in detail. To the best of our knowledge, the present study demonstrated for first time that stimulation of a glioblastoma multiforme (GBM) cell line (U87-MG) with hepatocyte growth factor (HGF) resulted in the phosphorylation of STAT5 at Tyr-694/699 and nuclear translocation of STAT5. In addition, HGF promoted nuclear translocation of STAT5 in a time-dependent manner and increased the proliferation of U87-MG cells. In order to determine the role of STAT5 directly, RNA interference was used to knockdown STAT5 expression in the U87-MG cell line. It was illustrated that small interfering RNA (siRNA) against STAT5 successfully inhibited the protein expression of STAT5 in the U87-MG cell line, leading to a potent suppression of tumor cell proliferation with or without HGF treatment. In order to broaden the investigation and to determine the role of STAT5 in vivo, immunohistochemistry (IHC) was applied to evaluate STAT5 expression in 100 newly diagnosed glioma and 10 non-neoplastic brain tissues. p-STAT5 expression increased according to the histopathological grade of the glioma. However, no p-STAT5 staining was observed in non-neoplastic brain tissues. These findings suggested that inhibition of the STAT5 pathway may be an effective therapeutic strategy for the clinical management of GBM.