〔Abstract〕 Objective To investigate the effect and underlying mechanisms of different culture conditions including growth status of parent culture and cell density on U251 cell proliferation and apoptosis. Methods Cell proliferation assay was applied to determine the growth of U251 cells under different culture conditions including growth status of parent culture and cell reseeding density. Western blot analysis was used to test the expressions of p-ERK1/2,cleaved-caspase3,cleaved-caspase8,p27,cyclin D1,p-PLK,p-FPAK. Caspase 3 activity assay was used to test cell apoptosis. Flow cytometry was performed for cell cycle distribution and proliferation. Results Re-plating of plateau phase U251 cells at high density resulted in increased proliferation. Re-plating of exponentially growing cells at high density resulted in limited proliferation accompanied by cell detachment. EH cells showed activated cleaved-Caspase 3 and cleaved-Caspase 8 as well as increased activity of Caspase 3, suggesting EH cells underwent apoptosis. Moreover, EH cells were accumulated in G2 phase by flow cytometry. Cyclin D1 and p-PLK1 were down-regulated while p27 expression was up-regulated in EH cells measured by Western blot. Increasing FAK expression did not rescue cells from apoptosis. Functional p27 expression prevented cells from undergoing apoptosis and kept cell proliferation. Conclusion The growth status of parent culture and cell density have impacts on U251 cell proliferation and apoptosis. Functional p27 expression plays an important role in the proliferation and survive of glioma.