The adoptive transfer of in vitro expanded Treg is a promising treatment option for autoimmune as well as alloantigen‐induced diseases. Yet, concerns about the phenotypic and functional stability of Tregs upon in vitro culture command both careful selection of the starting population and thorough characterization of the final cell product. Recently, a high degree of developmental plasticity has been described for murine Treg and Th17 cells. Similarly, IL‐17‐producing FOXP3⁺ cells have been detected among the CD45RA⁻ memory‐type subpopulation of human Tregs ex vivo. This prompted us to investigate the predisposition of human naïve and memory Tregs to develop into Th17 cells during polyclonal in vitro expansion. Here, we show that stimulation‐induced DNA demethylation of RORC, which encodes the lineage‐defining transcription factor for Th17 cells, occurs selectively in CD45RA⁻ memory‐type Tregs, irrespective of their FOXP3 expression level. On the contrary, naïve CD45RA⁺ Tregs retain stable CpG methylation across the RORC locus even upon prolonged ex vivo expansion and in consequence show only a marginal tendency to express RORC and develop into IL‐17‐producing cells. These findings are highly relevant for the generation of therapeutic Treg products.