BACKGROUND: The uneven use of synonymous codons in the transcriptome regulates the efficiency and fidelity of protein translation rates. Yet, the importance of this codon bias in regulating cell state-specific expression programmes is currently debated. Here, we ask whether different codon usage controls gene expression programmes in self-renewing and differentiating embryonic stem cells. RESULTS: Using ribosome and transcriptome profiling, we identify distinct codon signatures during human embryonic stem cell differentiation. We find that cell state-specific codon bias is determined by the guanine-cytosine (GC) content of differentially expressed genes. By measuring the codon frequencies at the ribosome active sites interacting with transfer RNAs (tRNA), we further discover that self-renewing cells optimize translation of codons that depend on the inosine tRNA modification in the anticodon wobble position. Accordingly, inosine levels are highest in human pluripotent embryonic stem cells. This effect is conserved in mice and is independent of the differentiation stimulus. CONCLUSIONS: We show that GC content influences cell state-specific mRNA levels, and we reveal how translational mechanisms based on tRNA modifications change codon usage in embryonic stem cells.