Objective:To optimize the culture conditions of glioma U87 cells for proliferation inhibiting assay. Methods:After down-regulation of HDGF expression by RNA interference in U87 cell line,cells were inoculated in 96-well plate. MTS cell proliferation assay was performed under three different conditions including serum free culture condition,containing 10% FBS culture condition and plate precoated with Matrigel. Results:U87 cell proliferation was inhibited after down-regulation of HDGF level. Cell proliferation inhibiting rates in serum free group,10% FBS group and Matrigel precoated group were 18%,9%,28%,respectively. Conclusion:Pre-coating the plate with Matrigel may be optimal for cell proliferation inhibiting detection after RNA interference.