Within the population of regulatory T cells (Tregs) natural Tregs (nTregs) and inducible Tregs (iTregs) can be distinguished. Although information about Tregs in swine exists, porcine iTregs were not under investigation yet. In this study, Foxp3(+) iTregs were generated from CD4(+)Foxp3(-) T cells by in vitro stimulation in the presence of IL-2 and TGF-β. In comparison to ex vivo Tregs these iTregs had a similar suppressive capacity on the proliferation of CD3-stimulated PBMC, caused higher levels of IL-10 in PBMC/Treg co-cultures, but did not suppress IFN-γ levels. The Ikaros family member Helios is currently discussed to distinguish iTregs and nTregs or to serve as an activation marker of Tregs. In this study, we demonstrate the cross-reactivity of an anti-mouse/human Helios mAb with porcine Helios. Flow cytometric analyses with this antibody showed that porcine iTregs do not express Helios after in vitro iTreg induction. Nevertheless, thymic Foxp3(+) T cells, which arise at the CD4/CD8α single-positive stage of T-cell development and are defined as nTregs, entirely expressed Helios. Although this might suggest the suitability of Helios as an nTreg-iTreg differentiation marker we also found that Helios(-) Tregs displayed a phenotype of naive CD4(+) T cells in vivo. Since iTregs are by definition activated/differentiated Tregs, this finding precludes that all Helios(-) Tregs are iTregs and thus also the use of Helios as a selection marker for porcine nTregs. Furthermore, Helios(+) Tregs displayed a more differentiated phenotype indicating that Helios might rather serve as a Treg activation/differentiation marker.