To investigate the effect of Kozak sequence (+4A or +4G) on expression of green fluorescent protein (GFP) gene in HEK293 cells. The eukaryotic expression vectors containing GFP gene with different Kozak sequence (+4A or +4G) were constructed by classic DNA recombination methods, including PCR, enzyme digestion, ligation, transformation, identification, et al. Two different Kozak sequences (+4A or +4G) were obtained through PCR with different mutagenic primers. The right recombinant plasmids pHGFP-A and pHGFP-G were transfected into HEK293 cells by liposome-mediated gene transfer method. The expression level of GFP was observed by fluorescent microscope, flow cytometry and Western blot. The flow cytometry revealed that the expression levels of GFP fluorescence in pHGFP-A and pHGFP-G transfected cells were about 15% and 45%, respectively. Western blot showed the specific bands of about 27 kD (GFP) both in pHGFP-G and pHGFP-A sample lanes; and the GFP expression density of pHGFP-G was about 3.87-fold as that of pHGFP-A by ImageJ software analysis. These results indicated that the +4G in Kozak sequence (when ?3 site is purine base pair) plays an important role in GFP protein translation, which enhances the GFP expression up to 4-fold in HEK293 cells.