Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN). Here, using Chinese hamster ovary (CHO) Niemann-Pick type C1 (NPC1) mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6) accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs). This increases Stx6/VAMP3 interaction and interferes with the recycling of αVβ3 and α5β1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion. Display omitted •Late endosomes determine TGN cholesterol levels to regulate Stx6 location•Cholesterol regulates Stx6 complex formation with VAMP3 and VAMP4•Stx6 mislocation in recycling endosomes (REs) inhibits the recycling of integrins•Stx6 accumulation in REs reduces cell migration and invasion Cell migration is fundamental to many physiological and pathological settings such as wound healing and cancer cell metastasis. Here, Reverter et al. show that the intracellular cholesterol levels determine the capacity of cells to initiate cell migration, adhere, and/or invade. Fine-tuning of cholesterol levels at Golgi/endosome boundaries is critical for regulating the trafficking and function of specific SNARE proteins that eventually regulate integrin recycling and cell behavior.