Synonymous mutations in protein coding genes significantly impact translation efficiency. We synthesized a pair of genes encoding green fluorescent protein that were separated by 160 synonymous mutations to investigate key factors that affect translation efficiency. One sequence was optimized for Escherichia coli (GFP(Eco)) and the other for Bacillus subtilis (GFP(Bsu)). When the genes were expressed in E coli, GFP(Eco) fluoresced 12-fold stronger than GFP(Bsu), confirming the suboptimal nature of the GFPBsu gene. We then employed directed evolution to improve the expression of GFPBsu. Random mutagenesis and DNA shuffling was used to generate mutant libraries, which were screened for fluorescence. A variant showing 6-fold fluorescence enhancement was identified, which contained a single mutation (G10A) in a rare codon for Gly-4. However, the substitution generated another type of rare codon, AGA, for Arg, suggesting that the improvement was caused by a factor other than the rare codon. We next applied saturation mutagenesis to Gly-4. The darkest variant contained a GGG codon (GFP(Bsu)-G) for Gly-4. Taking the location of the mutation into account, we hypothesized that destabilization of the mRNA secondary structure around the initiation codon improved the expression. We then randomized the nucleotide triplet in 5'-untranslated region (5'UTR) of GFP(Bsu), which is complementary to the Gly-4 codon. A variant showing 6-fold fluorescence enhancement was identified, which exhibited a destabilized secondary structure. When this 5'UTR sequence was combined with GEP(Bsu)-G, 22-fold fluorescent improvement was achieved. Collectively, the stability of the mRNA secondary structure around the initiation codon predominantly affected the translation efficiency.