The IL-12p40 cytokine subunit is critical for host defence against mycobacteria. Macrophages are major host cells for mycobacteria and a key cellular source of IL-12p40. Toll-like receptor 2 (TLR2) is a central driver of mycobacteria-induced IL-12p40 production. TLR2 heterodimerises with TLR1 or TLR6 to facilitate cellular activation, and both co-receptors contribute to host recognition of mycobacteria. Which TLR2 co-receptor interactions are required for mycobacteria-induced macrophage IL-12p40 production is unknown. Using primary murine macrophages, we confirmed requirement of TLR2 for macrophage IL-12p40 responses to Mycobacterium bovis BCG (BCG), the current tuberculosis vaccine. Contrary to expectations, neither TLR1 nor TLR6 were necessary for BCG-induced macrophage IL-12p40 responses. Instead, the TLR family member Radioprotective 105 (RP105) was required for BCG-induced IL-12p40 production in a manner dependent on TLR2. RP105 lacks the TLR intracellular Toll/IL-1 receptor domain essential for TLR signalling, which is consistent with lack of RP105 contributions to canonical TLR2 signalling. How RP105 contributes to mycobacteria-induced macrophage activation is unknown. RP105 contains a short cytoplasmic tail with evolutionary conserved amino acid residues of unknown function. Employing targeted mutagenesis and retroviral reconstitution of Rp105-deficient primary macrophages, we show that conserved amino acids in the RP105 cytoplasmic tail were essential for BCG-induced macrophage IL-12p40 production in a manner dependent on TLR2. Furthermore, biochemical analyses combined with high-resolution microscopy revealed that the RP105 cytoplasmic tail regulates RP105 subcellular distribution in macrophages and serves as a recruitment platform for intracellular trafficking, signalling and scaffolding machinery in BCG-infected macrophages. These findings uncovered, for the first time, a functional role for the RP105 cytoplasmic tail in the host recognition of mycobacteria. Collectively, our data highlight a key role for RP105 as the dominant TLR2 co-receptor in the recognition of mycobacteria and provide the basis for delineating unconventional TLR signalling events that orchestrate key host responses to mycobacterial infection.