A multimerization strategy to improve yields upon recombinant production of the 31-aa human proinsulin C-peptide is presented. Gene fragments encoding the C-peptide were assembled using specific head-to-tail multimerization. DNA constructs encoding one, three or seven copies of the C-peptide gene, fused to a serum albumin binding affinity tag, were expressed intracellularly in Escherichia coli. The three fusion proteins were produced at similar levels (approximately 50 mg/l) and were proteolytically stable during production. Enzymatic digestion by trypsin-carboxypeptidase B treatment of the fusion proteins was shown to efficiently release native C-peptide, as determined by mass spectrometry, reverse-phase chromatography and a radioimmunoassay. The quantitative yields of C-peptide obtained from the three different fusion proteins suggest that this multimerization strategy could provide a cost-efficient production scheme for the C-peptide, and that this strategy could be useful also for production of other recombinant peptides.