Although described in the 70’s, CD8+regulatory T cells (Tregs) remain incompletely understood and to date, although several markers are used to define them, they remain poorly defined. The identification of reliable and consistent markers, as it was done for CD4+Tregs, remains an urgent task and a challenge to advance our understanding. Herein, we analyzed total CD8+T cells using single cell CITEseq and VDJ T cell receptor sequencing utilizing markers used previously to identify Tregs, in particular CD45RC described by our team and others to divide pro-inflammatory (CD45RChigh) and pro-regulatory (CD45RClow/-) CD8+T cells in rat, mice and human. 7000 freshly isolated, non-stimulated CD8+T lymphocytes of four healthy volunteers were analyzed. Combining at a single cell level transcriptome and protein expression data led for the first time to the characterization and definition of three subsets of regulatory CD8+T cells. Furtherin vitrofunctional analysis based on three markers highlighted the superior suppressive activity of the CD8+CD45RClow/-TNFR2+CD29lowTregs subset.To our knowledge, this is the largest characterization of human CD8+Tregs to date. This data resource will help improve our understanding of CD8+T cells heterogeneity and will help to translate CD8+Tregs to the clinic.