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Objective: The purpose of this study was to determine the effects of IGFBP-3 and GalNAc-T14 on the proliferation and cell cycle of glioblastoma cells and to explore the mechanisms of action., Methods: U87MG and U251MG glioblastoma cells were treated with recombinant human IGFBP-3 (rhIGFBP-3). Furthermore, IGFBP-3-overexpressed cells and cells co-overexpressing IGFBP-3 and GalNAc-T14 were constructed by transfection. Cell viability, cell colony formation ability, cell cycle and protein expression were determined by MTT assay, colony formation assay, flow cytometry and Western blotting, respectively., Key Findings: Both rhIGFBP-3 treatment and overexpression of IGFBP-3 induced the proliferation, colony formation, and G1/S phase transformation of U87MG and U251MG cells. In addition, the expression of cyclinE, CDK2 and p-ERK1/2 proteins was up-regulated in the cells. In cells co-overexpressing, IGFBP-3 and GalNAc-T14, cell proliferation, colony formation and G1/S phase transformation were inhibited, and the expression of CyclinE, CDK2 and p-ERK1/2 was significantly down-regulated, when compared with IGFBP-3-overexpressed cells., Conclusions: IGFBP-3 can promote the proliferation, colony formation and G1/S phase transformation of U87MG and U251MG cells, which may be related to the activation of ERK signalling pathway and the up-regulation of cyclinE and CDK2 proteins. Furthermore, our study demonstrated that GalNAc-T14 can inhibit the functions of IGFBP-3., (© 2019 Royal Pharmaceutical Society.)