BACKGROUND Idiopathic pulmonary fibrosis (IPF) is a fatal progressive interstitial pneumonia. The innate immune system provides a crucial function in the recognition of tissue injury and infection. Toll-like receptor 3 (TLR3) is an innate immune system receptor. We investigated the role of a functional TLR3 single-nucleotide polymorphism in IPF. OBJECTIVE To characterize the effects of the TLR3 Leu412Phe polymorphism in primary pulmonary fibroblasts from patients with IPF and disease progression in two independent IPF patient cohorts. To investigate the role of TLR3 in a murine model of pulmonary fibrosis. METHODS TLR3-mediated cytokine, type 1 IFN, and fibroproliferative responses were examined in TLR3 wild-type (Leu/Leu), heterozygote (Leu/Phe), and homozygote (Phe/Phe) primary IPF pulmonary fibroblasts by ELISA, real-time polymerase chain reaction, and proliferation assays. A murine model of bleomycin-induced pulmonary fibrosis was used in TLR3 wild-type (tlr3(+/+)) and TLR3 knockout mice (tlr3(-/-)). A genotyping approach was used to investigate the role of the TLR3 L412F polymorphism in disease progression in IPF using survival analysis and longitudinal decline in FVC. RESULTS Activation of TLR3 in primary lung fibroblasts from TLR3 L412F-variant patients with IPF resulted in defective cytokine, type I IFN, and fibroproliferative responses. We demonstrate increased collagen and profibrotic cytokines in TLR3 knockout mice (tlr3(-/-)) compared with wild-type mice (tlr3(+/+)). TLR3 L412F was also associated with a significantly greater risk of mortality and an accelerated decline in FVC in patients with IPF. CONCLUSIONS This study reveals the crucial role of defective TLR3 function in promoting progressive IPF.