The mammalian proton coupled transporter (POT) hPepT1 has been studied intensively due to its role in nutrient and drug absorption in the small intestine. In the absence of a crystal structure of hPepT1, the available structures of bacterial POTs, among which PepTSo from Shewanella oneidensis has a strikingly high sequence identity, can be used to rationalize its mechanism and substrate preference. However, very little is known about the substrate specificity of PepTSo. To elaborate on this, the natural peptide specificity of PepTSo was investigated. Di and tri-peptides were found to be substrates for PepTSo in contrast to mono- and tetrapeptides as was indicated by previous competition studies. Interestingly, a negatively charged side chain was better accommodated on the dipeptide N- than the C-terminus position. Inversely, a positive charged side chain appeared to be tolerated better on the dipeptide C- than the N-terminus. Although the results on chain length preference are consistent with the previously published results on hPePT1, the selectivity of charge is opposite and something which may be explained by the presence of a non-conserved lysine 318 in the PepTSo active site. The preference for a negatively charged side chain in the N-terminus was also observed for tripeptides. Recent literature has discussed the tripeptide orientation based on ambiguous crystal structures. The data presented here strongly indicates that di- and tripeptides bind in a similar orientation.