Clonorchis sinensisis a zoonotic parasite associated with liver fibrosis and cholangiocarcinoma development. The role of toll-like receptors (TLRs) inC.sinensisinfection has not yet been fully elucidated. Here, the TLR3 signaling pathway, cytokine expression and liver fibrosis were examined inC.sinensis-infected wildtype (WT) andTLR3-/-mice. Polyinosinic-polycytidylic acid (Poly (I:C)) was used to treatC.sinensisinfections. The results showed that TLR3 deficiency caused severe clonorchiasis with increased parasite burden, exacerbated proinflammatory cytokine expression and liver lesions, promoted the TGF-β1/Smad2/3 pathway and myofibroblast activation, exacerbated liver fibrosis (compared to WT mice). Poly (I:C) intervention increased the body weight, decreased mouse mortality and parasite burden, reduced liver inflammation, and alleviatedC.sinensis-induced liver fibrosis. Furthermore,C.sinensisextracellular vesicles (CsEVs) promote the production of IL-6, TNF in WT biliary epithelial cells (BECs) via p38/ERK pathway, compared with control group, while TLR3 deletion induced much higher levels of IL-6 and TNF inTLR3-/-BECs than that in WT BECs. Taken together, TLR3 inhibit IL-6 and TNF production via p38/ERK signaling pathway, a phenomenon that resulted in the alleviation ofC.sinensis-induced liver fibrosis. Poly (I:C) is a potential treatment for clonorchiasis.