We previously demonstrated that the formation of hypertrophic scarring on the wounds of a rabbit ear fibrotic model was significantly reduced by grafting a bilayer skin substitute expressing indoleamine 2,3-dioxygenase (IDO). Here, we hypothesize that the improved healing quality is due to extracellular matrix modulatory effect of IDO-mediated tryptophan metabolites. To test this hypothesis, a series of in vitro and in vivo experiments were conducted and the findings revealed a significant increase in the expression of matrix metalloproteinase 1 (MMP-1) in fibroblasts either transduced with human IDO gene or cultured with conditioned media obtained from IDO-expressing cells. Consistent with this finding, kynurenine (Kyn) treatment markedly increased the levels of MMP-1 and MMP-3 expression through activation of the MEK (mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase)-ERK1/2 MAPK signaling pathway. On the other hand, Kyn significantly suppressed the expression of type I collagen in fibroblasts as compared with that of control. To test the anti-fibrogenic effect of Kyn in an in vivo model, rabbit ear fibrotic wounds were topically treated with cream containing 50 μg Kyn per l00 μl of cream per wound. The result showed a marked improvement in scar formation relative to the controls. These findings collectively suggest that Kyn can potentially be used as an anti-fibrogenic agent for treating hypertrophic scarring.