Nucleic acid-sensing Toll-like receptors (TLRs) are subject to complex regulation to facilitate recognition of microbial DNA and RNA while limiting recognition of self-nucleic acids. Failure to properly regulate these TLRs can lead to autoimmune and autoinflammatory disease. Intracellular localization of these receptors is thought to be critical for self versus non-self discrimination, yet the molecular mechanisms that reinforce compartmentalized activation of intracellular TLRs remain poorly understood. Here we describe a new mechanism that prevents TLR9 activation from locations other than endosomes. This control is achieved through the regulated release of the receptor from its trafficking chaperone UNC93B1, which occurs only within endosomes and is required for ligand binding and signal transduction. Preventing TLR9 release from UNC93B1, either through mutations in UNC93B1 that increase affinity for TLR9 or through an artificial tether that impairs release, results in defective signalling. While TLR9 and TLR3 release from UNC93B1, TLR7 does not dissociate from UNC93B1 in endosomes and is regulated via distinct mechanisms. This work defines a new checkpoint that reinforces compartmentalized activation of TLR9 and provides a mechanism by which activation of individual endosomal TLRs may be distinctly regulated.