Adipose-derived stem cells (ASCs) are emerging therapeutics in immunomodulation. Short-chain fatty acids, particularly butyrate, regulate immune responses and adipose tissue (AT) function. However, the effects of butyrate on ASCs remain unclear. We hypothesize that butyrate modulates the secretory factors of ASCs to reinforce their immunosuppression. Microarray analysis was performed to determine the secretory immunoregulatory factors of murine ASCs that are augmented by butyrate. The secretion of the corresponding proteins was confirmed for the selected factors. We demonstrated that in the concomitant presence of butyrate and ASCs, the inhibition of activated splenocyte proliferation in vitro was pronounced. Butyrate increased the percentages of ASC-induced Foxp3+ regulatory T cells and further reduced ASC-suppressed T helper 17 cells. The enhanced immunosuppressive properties of ASCs generated under the influence of butyrate is independent of prostaglandin E2, interleukin-6 and galectin-1. ASCs cocultured with activated splenocytes plus butyrate exhibited higher gene expression for inducible nitric oxide (NO) synthase (iNos), chemokines, placental growth factor, and amphiregulin (Areg) than ASCs cocultured with activated splenocytes alone did. Mechanistically, butyrate regulated the elaboration of iNOS-inducing cytokines, IFN-γ and TNF-α, in the ASC–splenocyte coculture, triggering ASCs to produce high levels of NO. Blocking assays confirmed that AREG and iNOS non-redundantly regulated and augmented ASC immunosuppression in the presence of butyrate. Collectively, our findings provided a previously unrecognized mechanism underlying immunomodulation involving coupling through butyrate and ASCs. Additional studies to prevent and counteract AT inflammation and its associated disorders by modulating ASC immunomodulation with butyrate are thus recommended.