Cell-penetrating peptides are molecules with the ability to cross membranes and enter cells. Attention has been put on these peptides as a tool for drug delivery research, as they are able to serve as delivery vectors for large molecules. Intracellular delivery of bioactive peptides is a very promising research area for clinical applications, since peptides are able to simulate protein regions and thus modulate key intracellular protein-protein interactions. Therefore, evaluation of different strategies for production of these peptides is necessary. In this work, an intein-mediated system was used to evaluate Escherichia coli recombinant production of p53pAnt and PNC27 anticancer cell-penetrating peptides. It was demonstrated that the pTXB1 and the pTYB11 vector systems are suitable for production of this kind of peptides. The production process involves a low-temperature induction process and an efficient on-column intein-mediated cleavage, which allowed an effective peptide recovery using a single chromatographic step.