Abstract The role of B cells in tumor infiltrations is controversially discussed. Different studies suggest that certain tumor-infiltrating B cell populations exhibit regulatory potential. Here, we demonstrate that the microenvironment of various solid tumors contains granzyme B (GrB)-expressing B cells adjacent to IL-21-providing T cells. Since GrB-mediated effector T cell modulation is known from regulatory T cells (Treg) and plasmacytoid dendritic cells, we hypothesized the existence of similar mechanisms in B cells. Here we show that IL-21 induces B cells expressing high levels of GrB and controlling T cell proliferation by GrB-dependent degradation of the T cell receptor ζ-chain. Detailed characterization of IL-21-induced GrB+ B cells reveals a CD19+CD38+CD1d+IgM+CD147+ phenotype and expression of additional regulatory molecules including IL-10, CD25 and IDO. Of note, IL-21-mediated GrB induction integrates both BCR- and TLR-mediated signals and is enhanced in the presence of B cell CD5 expression. This is the first report demonstrating that IL-21 induces GrB+ human regulatory B cells, which can be detected in tumor infiltrations, and which may contribute to the modulation of cellular adaptive immune responses by Treg-like mechanisms. Our findings may stimulate the development of novel diagnostic and cell therapeutic approaches to the management of malignant, autoimmune and graft-versus-host pathologies.