Short-chain fatty acids (SCFAs) have been recognized as mediators of immune responses, including pathways of cytokine production. In this study, we investigated the immune-regulatory effects of SCFAs on human peripheral blood mononuclear cells (PBMCs) from buffy coat of healthy donors. PBMCs were exposed to varying concentrations of individual SCFAs or of their mixtures of acetate, propionate and butyrate. The productions of interleukin (IL) IL-1β, IL-2, IL-6, IL-10, IL-17, IL-21, IL-23 and transforming growth factor beta 1 (TGF-β1) were assessed. T cell differentiation after exposure to SCFAs was also examined. Compared with lipopolysaccharide (LPS)-stimulated cells (controls), SCFAs slightly decreased TGF-β1 production and reduced IL-6 production; butyrate was more effective than acetate or propionate. SCFAs particularly butyrate caused the induction of CD4+CD25+ regulatory T cells (Treg) rather than Th17 cells. SCFAs may up-regulate the production of anti-inflammatory cytokines in PBMCs, resulting in the induction of CD4+CD25+ Treg cells.