The objective of this study was to uncover distinct cellular and genetic signatures of transplant operational tolerance (TOT) in kidney transplant recipients (KTRs) through single cell RNA sequencing (scRNA-seq) using peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from 12 KTRs, including those with TOT (TOT, n = 4), stable allograft function on maintenance immunosuppression (STA, n = 4) and biopsy-proven allograft rejection (BPAR, n = 4). ScRNA-seq of PBMCs was analyzed using 20 cell surface marker antibody sequencing to annotate clusters and 399 immune response panel to identify gene expression. Differences in cellular distribution and gene expression were compared among the three groups. Heatmap hierarchical clustering showed that overall cellular distribution pattern was distinct in TOT in comparison with those in the other two groups, with the proportion of B cells being higher in TOT, attributed to immature B cell fraction (TOT vs. STA vs. BPAR: 4.61% vs. 1.27% vs. 2.53%, p = 0.01). Transcript analysis of B cells revealed that genes involved in allo-immune pathway were downregulated in TOT. In T cell subset analysis, the proportion of naïve T cells and regulatory T cells (Tregs) was increased. In transcript analysis, genes associated with inflammation were decreased, while expression levels of CCR6 in Tregs were increased in TOT. Proportions of NKT and NK cells were increased in TOT than in the other two groups. This study showed that TOT has distinct cellular and genetic signatures such as increases of immature B cells, naïve T cells and Tregs and high expression levels of CCR6 in Tregs. Keywords: CCR6; immune tolerance; kidney transplantation; peripheral blood mononuclear cells; single-cell analysis.