TLR4, a member of the Toll-like receptor (TLR) family, activates host cells in response to lipopolysaccharide (LPS). Endocytosis of TLR4 is a significant regulatory step in LPS-driven cellular activation, curbing MyD88-mediated pro-inflammatory cytokine responses in favour of TRIF-dependent endosomal signalling and Type I interferon (IFN) expression. Radioprotective 105 kDa (RP105) is a TLR family member expressed by macrophages, myeloid dendritic cells (DCs) and B cells. Previous studies have reported elevated LPS-induced pro-inflammatory cytokine production in RP105-deficient (Rp105-/-) macrophages and DCs, leading to the conclusion that RP105 negatively regulates TLR4-driven pro-inflammatory cytokine responses in myeloid cells. These findings contradict the previously described positive regulatory role of RP105 in LPS-induced B cell proliferation. Here we demonstrate that Rp105-/- primary mouse macrophages and CRISPR/Cas9-derived RP105-negative RAW264.7 macrophages exhibit impaired LPS-induced TLR4 internalisation, TBK-1 phosphorylation, and IFN-β expression. In addition, Rp105-/- mice exhibited significantly reduced serum IFN-β relative to WT mice following LPS challenge. Use of small molecule inhibitors indicated that the molecular mechanisms underlying RP105-mediated TLR4 internalisation differ from those currently implicated as effectors of CD14- and DAP12/FcRγ-driven TLR4 internalisation. Together, these data identify RP105 as a previously unidentified positive regulator of LPS-driven TLR4 internalisation and Type I IFN expression, and further elucidate molecular mechanisms by which RP105 shapes the host response to LPS.