Cellular rejection of xenografts is predominantly mediated by CD4⁺ T cells. Human CD4⁺CD25⁺ regulatory T cells (Tregs) are capable of suppressing the CD4⁺ T cell‐mediated xenogeneic response in vitro. However, the precise mechanisms that are involved remain to be identified. In this study, we analyzed whether interleukin‐10 (IL‐10) is required for Tregs to suppress xenogeneic responses in vitro by small interfering RNA (siRNA)‐mediated IL‐10 knockdown. After siRNA transfection, Tregs were analyzed for IL‐10 gene and protein expression and their phenotype. Mixed lymphocyte reactions (MLRs) were performed by stimulating human CD4⁺CD25⁻ T cells with allogeneic or pig peripheral blood mononuclear cells (PBMCs) in the presence or absence of Tregs in a coculture or transwell system. The production of effector cytokines by xeno‐ or alloreactive CD4⁺CD25⁻ T cells, or suppressive cytokines by Tregs, was examined using enzyme‐linked immunosorbant assay (ELISA). We showed that IL‐10 knockdown resulted in a substantially reduced IL‐10 production by Tregs, leading to impaired Treg‐mediated suppression of xeno‐ but not alloreactive CD4⁺ CD25⁻ T‐cell proliferation. However, IL‐10 knockdown had no effect on Treg phenotype, their suppression of effector cytokine production by xeno‐ or alloreactive T cells and the production of the Treg‐suppressive cytokine, transforming growth factor‐β (TGF‐β). This study shows that IL‐10 is required for human Tregs to suppress xenogeneic but not allogeneic proliferation in vitro.