Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate the mechanisms, we engineered a synthetic library of 154 genes that vary randomly at synonymous sites, but all encode the same green fluorescent protein. When expressed in E. coli, GFP protein levels varied 250-fold across the library. GFP mRNA levels, mRNA degradation patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near the ribosomal binding site explained over half the variation in protein levels. In our analysis, mRNA folding and associated rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.