Polyriboinosinic-polyribocytidylic acid (polyI:C) is a viral dsRNA analoguethat promotes wounds healing, accelerates re-epithelialization, granulation and neovascularization, and induces pro-inflammatory cytokine release. Little is known about polyI:C mediated induction of inflammatory mediators in human dermal fibroblast (HDFs), which form the primary scaffold for epithelial cells covering the wound. Here, we found that polyI:C enhances IL-6 and IL-8 mRNA expression and induces of IL-6 and IL-8 production in a concentration-dependent and time-dependent manner in HDFs. PolyI:C treatment rapidly increased phosphorylation level of both STAT3 and p38 mitogen-activated protein kinase (MAPK). Moreover, pretreatment with AG490, a Janus kinase (JAK) inhibitor, inhibited polyI:C-induced STAT3 phosphorylation and subsequent IL-6 and IL-8 release. Conversely, pretreatment with SB203580, a selective inhibitor of p38 MAPK, blocked p38 MAPK phosphorylation and IL-6 and IL-8 expression. In conclusion, polyI:C induces IL-6 and IL-8 production in HDFs via the JAK/STAT3 and p38 MAPK signaling pathways.