Toll-like receptors (TLRs) are germline-encoded pattern-recognition receptors that initiate innate immune responses by recognising molecular structures shared by a wide range of pathogens, known as pathogen-associated molecular patterns (PAMPs). TIR domain-containing adaptor inducing IFN-β (TRIF) is an important adaptor protein in TLR3 and TLR4 signalling pathways that mediate proinflammatory cytokine and IFN responses following their activation by doubled-stranded RNA and LPS, respectively. In the present study, we sought to investigate the novel proteins and pathways that serve to modulate the functionality of TRIF. To this end, immunoprecipitation and LC-MS analyses of the overexpressed and endogenous TRIF immunocomplexes were performed. A disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), Segment polarity protein dishevelled homolog DVL3 (DVL3) and Optineurin (OPTN) were identified as TRIF-interacting partners. Their interactions with TRIF were also confirmed by direct immunoprecipitation experiments of overexpressed and endogenous TRIF. More specifically, ADAM15 was found to interact with TRIF at 60 and 20 min. upon poly(I:C) and LPS stimulation, respectively. Overexpression of ADAM15 in HEKT293, HEK293-TLR3 and HEK293-TLR4 inhibited TRIF, TLR3 and TLR4-dependent activation of NF-κB, IFNβ and Rantes promoter activation, respectively. Moreover, downregulation of ADAM15 by esiRNA enhanced poly(I:C) and LPS-induced cytokine/chemokine secretion in the U373-CD14 cell line. This study also demonstrated that all of the three DVLs isoforms (DVL1, DVL2 and DVL3) interacted constitutively with TRIF when overexpressed in HEK293-TLR4. However, immunoprecipitation of endogenous TRIF in U373-CD14 cell line showed that DVL3 associated with TRIF at 20-40 min upon LPS, but not poly(I:C) stimulation. Overexpression of all DVLs isoforms in HEKT293 and HEK293-TLR3 decreased TRIF and TLR3-induced NF-κB, IFNβ and Rantes, respectively. Nevertheless, DVLs inhibition using a chemical inhibitor attenuated poly(I:C) and LPS-dependent upregulation of TNFα, IFNβ and Rantes mRNA, as well as LPS-induced phosphorylation of IRF3 in wild-type murine BMDMs. Furthermore, the study also showed that OPTN interacted constitutively with TRIF when overexpressed in HEK293-TLR3. Immunoprecipitation of endogenous TRIF revealed that OPTN interacted with TRIF in a ligand-dependent manner only. Poly(I:C) and LPS stimulation in U373-CD14 increased OPTN protein expression levels after 24 h. Overexpression of OPTN negatively regulates TRIF and TLR3-dependent reporter gene activation. In addition, suppression of OPTN expression using esiRNA increased poly(I:C)-induced IFNβ mRNA expression as well as TNFα, and Rantes secretion in U373-CD14. However, suppression of OPTN decreased/increased LPS-induced TNFα/Rantes, respectively. In conclusion, this study analysed, for the first time, TRIF immunocomplex and identified ADAM15, DVL3 and OPTN as novel TRIF interacting proteins and showed their role in TRIF-mediated TLR signalling. This study has advanced our understanding of the complexities of TRIF signalling in the context of TLR and we proposed that TRIF could be a key modulator of alternate signalling pathways.