Objective To construct a eukaryotic expression vector for MEK2-siRNA to explore the expression of this endogenous MEK2 in glioma U87 cell line. Methods Four single-stranded template DNAs encoding siRNA against MEK2, each consisting of 60 bp, were synthesized chemically;based on these 4 single-stranded template DNAs, 2 double-stranded DNAs were formed by annealing,then identified by restriction analysis and inserted into vector pSUPER.basic by T4 ligase. Positive recombinants were indentified by EcoRI and HindⅢ double digestion and transfected into the U87 cells.The protein expression level of MEK2 was determined by using Western blotting. Results Both restriction analysis and sequencing proved that the eukaryotic expression vector for MEK2-siRNA was constructed correctly. MEK2-siRNA plasmid screened out by Western blotting. The MEK2 expression level in negative control group was (0.105±0.023) and that in transfected group was (0.030±0.006).Conclusion The eukaryotic expression vector for MEK2-siRNA is successfully constructed, which down-regulates the transcription of MEK2 protein in U87 cells. It provides a certain experimental basis for gene therapy of glioma by RNAi technique.