Objective  To investigate the effect of methotrexate (MTX) on the localization of RAS protein and the downstream signal pathway and to explore the mechanism of MTX to inhibit the growth of human glioblastoma cell line U87. Methods  After the treatment of U87 cells with different concentrations of MTX (75nmol/L, 375nmol/L, 750nmol/L, 7,500nmol/L, 15,000nmol/ L), the cell viability and apoptosis rate were detected by MTT and FCM respectively. The distribution of RAS protein and activation was observed by confocal microscopy and the expression level of the MAPK/ERK protein in the downstream was measured by immune blotting method after U87 cell transduced retrovirus carrying RAS-GFP fusion gene. Further, the effect of MTX on the level of phosphorylation of P-p42/p44 MARK protein and the level of its regulation of the transcription factor c-MYC expression using different MAKP/ERK signaling pathway specific inhibitors (U0126 and PD98059) was investigated. The expression level of CD47 on the surface of cells was further assayed by FCM on U87 cells treated with low dose of MTX at different time points. Results  The different concentrations of MTX (75nmol/L, 375nmol/L, 750nmol/L, 7,500nmol/L, 15,000nmol/L) could inhibit the proliferation of human glioblastoma cell line U87 in a dose-dependent manner but can not directly induce apoptosis. The cell cycle of most U87 treated with the different concentrations of MTX was arrested at G2/S compared to untreated U87 cells. After U87 cells transduced RAS-GFP fusion gene (100% GFP) were treated with U87compared with the control group, the RAS protein was mislocation from the cell membrane to the cytoplasm; the P-p42/p44 MARK level was inhibited (P<0.05); the expression level of the transcription factor c-MYC was down-regulated (P<0.05). More important we addressed that CD47 related to glioblastoma growth and metastasis on the cell surface of U87 treated with MTX was decreased at different times, which confirmed by flow cytometry. Conclusions  It is the first time that we have demonstrated that MTX can inhibit the growth of glioblastoma cell line the U87; cell cycle was arrested at G2/S; the localization of the RAS protein was changed; RAS/MAPR/ERK/C-MYC signaling pathway was inhibited; MTX may be an alternative therapeutic drug for patients with recurrent glioblastoma.