Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have been proven to present immunosuppressive effects in both experimental and clinical trials which is targeting various rare diseases including inflammatory bowel disease (IBD). In addition, recent studies have reported tryptophanyl-tRNA synthetase (WRS) possess uncanonical roles such as angiostatic and anti-inflammatory effects. However, little is known about the function of WRS in MSC-based therapy. In present study, we investigated whether a novel factor, WRS, secreted from MSCs has a role in amelioration of human MSC against IBD and specific mechanism underlying MSC therapy. Experimental colitis was induced by administration of 3% DSS solution to 8-week-old mice and human umbilical cord blood-derived MSCs (hUCB-MSCs) were injected intraperitoneally. Secretion of WRS from hUCB-MSCs and direct effect of WRS on isolated CD4+ T cells was determined via in vitro experiments and hUCB-MSCs showed significant therapeutic rescue. Importantly, WRS level in the serum of colitis induced mice was decreased and recovered by the administration of MSCs. Through in vitro examination, WRS expression of hUCB-MSCs was increased when the cells were treated with interferon-γ (IFN-γ). Moreover, WRS itself was evaluated and shown to have a role in inhibiting activated T cells by inducing apoptosis. In summary, IFN-γ-mediated secretion of WRS from MSCs has a role in suppressive effect on excessive inflammation and disease progression of IBD and it brings new highlights in the immunomodulatory potency of hUCB-MSCs.