The function of the adaptor molecule MyD88 is thought to be independent of toll-like receptor 3 (TLR3) signaling. This study aimed to identify certain previously unknown roles of MyD88 in TLR3 signaling during the promotion of pro-inflammatory cytokine production. Upon conducting an analysis of all the TLR ligands, it was found that the TLR3-specific ligand polyinosinic: polycytidylic acid (Poly I:C) significantly induced the production of TNF proteins and up-regulation of other TLR transcripts, particularly TLR2. Accordingly, TLR3 stimulation also led to a significant up-regulation of the endogenous TLR2 ligands HMGB1 and Hsp60. In contrast, TLR3 silencing significantly down-regulated MyD88 and TLR2 expression, and pro-inflammatory IL-1β, TNF, and IL-8 cytokine secretion. The silencing of MyD88 similarly led to the down-regulation of TLR2, IL1β, TNF, and IL-8, which suggests that MyD88 was active downstream of TLR3. The animal model, i.e., the MyD88 knockout mouse presented with lower TNF, NF-κB , and IRF-3 levels, as compared to those in the control wild type mouse treated with Poly I:C. Taken together, our results demonstrate a previously unknown role of MyD88 in the TLR3 signaling pathway; this finding highlights the importance of TLRs and adapter protein interplay for the modulation of the endogenous TLR ligands involved in pro-inflammatory cytokine regulation.