Abstract Compartmentalization of nucleic acid (NA)–sensing TLR3, 7, 8, and 9 is strictly regulated to direct optimal response against microbial infection and evade recognition of host-derived NAs. Uncoordinated 93 homolog B1 (UNC93B1) is indispensable for trafficking of NA-sensing TLRs from the endoplasmic reticulum (ER) to endosomes/lysosomes. UNC93B1 controls loading of the TLRs into COPII vesicles to exit from the ER and traffics with the TLRs in the steady state. Ligand-induced translocation also happens on NA-sensing TLRs. However, the molecular mechanism for ligand-dependent trafficking of TLRs from the ER to endosomes/lysosomes remains unclear. In this study, we demonstrated that leucine-rich repeat containing protein (LRRC) 59, an ER membrane protein, participated in trafficking of NA-sensing TLRs from the ER. Knockdown of LRRC59 reduced TLR3-, 8-, and 9-mediated, but not TLR4-mediated, signaling. Upon ligand stimulation, LRRC59 associated with UNC93B1 in a TLR-independent manner, which required signals induced by ligand internalization. Endosomal localization of endogenous TLR3 was decreased by silencing of LRRC59, suggesting that LRRC59 promotes UNC93B1-mediated translocation of NA-sensing TLRs from the ER upon infection. These findings help us understand how NA-sensing TLRs control their proper distribution in the infection/inflammatory state.