Cell penetrating peptides (CPPs) have potential application as vectors to deliver hydrophilic molecules with therapeutic interest, which typically have very poor cell penetration. Recently in our lab we have identified disulfide-rich cyclic peptides able to internalize into mammalian cells, which were named cyclic cell penetrating peptides (cCPPs). Sunflower trypsin inhibitor-1 (SFTI-1), a 14-amino acid residue peptide obtained from sunflower seeds with potent trypsin-inhibitory activity, is a non-cytotoxic cCPP that can potentially be used as a scaffold to stabilize an active epitope and as a vehicle for drug delivery. The aim of this study is to characterize the internalization mechanism of SFTI-1, which is largely unknown. We have designed and synthesized a series of SFTI-1 mutants, including the enantiomer all-D-SFTI-1, to study the effect of structure on the internalization of SFTI-1. NMR spectra were recorded for all the synthesized mutants confirming that their tertiary conformation was essentially identical to that of native SFTI-1. Cytotoxicity studies showed that none of the mutants are toxic. Internalization studies with flow cytometry suggested that the cellular uptake is non-receptor dependent mechanism and also independent of the charge, cyclic backbone and disulfide bonds. Overall we found that SFTI-1 internalizes by a non-specific endocytic pathway. Together with a rigid backbone and stable structure, SFTI-1 is an attractive scaffold for drug design.