We studied the relationship between insertion of the last sense codon (i.e., the codon preceding the stop codon) and the efficiency of gene expression. We inserted 64 kinds of last sense codon at the 5’ end of the stop codon of the green fluorescent protein (GFP) gene and introduced the modified GFP genes into Escherichia coli (E. coli). Measuring the fluorescence intensity of the GFP produced in E. coli showed that the last sense codon influenced GFP gene expression and when CCG was inserted as the last sense codon, fluorescence intensity of E. coli was increased to 2.09 fold. On the other hand, insertion of CUA caused decrease of fluorescence intensity to 0.33 fold. We hope that our findings, which may be applicable to gene engineering, will be useful for further studies of protein expression.