The subcellular localization and intracellular trafficking of Toll-like receptors (TLRs) critically regulate TLRs-mediated antimicrobial immunity and autoimmunity. Here, it is demonstrated that the E3 ubiquitin ligase RNF115 inhibits the post-endoplasmic reticulum (ER) trafficking of TLRs and TLRs-mediated immune responses by catalyzing ubiquitination of the small GTPases RAB1A and RAB13. It is shown that the 14-3-3 chaperones bind to AKT1-phosphorylated RNF115 and facilitate RNF115 localizing on the ER and the Golgi apparatus. RNF115 interacts with RAB1A and RAB13 and catalyzes K11-linked ubiquitination on the Lys49 and Lys61 residues of RAB1A and on the Lys46 and Lys58 residues of RAB13, respectively. Such a modification impairs the recruitment of guanosine diphosphate (GDP) dissociation inhibitor 1 (GDI1) to RAB1A and RAB13, a prerequisite for the reactivation of RAB proteins. Consistently, knockdown of RAB1A and RAB13 in Rnf115+/+ and Rnf115-/- cells markedly inhibits the post-ER and the post-Golgi trafficking of TLRs, respectively. In addition, reconstitution of RAB1AK49/61R or RAB13K46/58R into Rnf115+/+ cells but not Rnf115-/- cells promotes the trafficking of TLRs from the ER to the Golgi apparatus and from the Golgi apparatus to the cell surface, respectively. These findings uncover a common and step-wise regulatory mechanism for the post-ER trafficking of TLRs