The initiation of effective host responses to a bacterial infection requires innate immune receptors that sense conserved microbial components and initiate intracellular signalling pathways that govern inflammation and antimicrobial defence. The Toll-like receptor (TLR) family member, TLR4, is pivotal in the host response to Gram-negative bacteria through the recognition of the outer membrane component lipopolysaccharide (LPS). Yet, inadequately controlled TLR4 activation contributes to systemic inflammation in severe manifestations of infection with Gram-negative bacteria. This emphasises the need to understand the mechanisms by which TLR4 is activated but also regulated and "turned off". Activation of TLR4 at the cell surface drives acute expression of pro-inflammatory cytokine responses; endosomal TLR4 promotes type I interferon expression while also sustaining pro-inflammatory signalling. Radioprotective 105 kDa (RP105) is a TLR family member previously reported as a negative regulator of TLR4 mediated pro-inflammatory cytokine responses to LPS. However, whether RP105 affects functions of endosomal TLR4 remains unknown. In this study, we position RP105 as a positive regulator of TLR4 functions in endosomes. Rp105-deficient macrophages exhibited decreased TLR4-mediated type I interferon expression as well as diminished phosphorylation of TANK-binding kinase 1 (TBK1) downstream of endosomal TLR4. Using confocal and high-resolution microscopy, we examined the intracellular distribution of RP105 and TLR4 and discovered that localisation of both receptors is dynamically regulated in response to LPS with differential distribution to distinct endosomal compartments. Our findings challenge the current view that RP105 inhibits TLR4 activation and position RP105 as a facilitator of TLR4 functions in endosomes. With these new insights, our data expand the understanding of the mechanisms that govern TLR4 functions. Such knowledge might be harnessed for practical applications that enable the selective activation or inhibition of distinct functional outputs of TLR4 from different subcellular localisations.