Cholesterol is considered indispensable for the functioning of integrins in cell migration. However, the physiological cholesterol pools that control integrin trafficking remain unclear. We have previously demonstrated that the Niemann Pick Type C1 (NPC1) mutation, which is characterized by an inhibition of cholesterol export from late endosomes (LE), interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface for cell migration (Cell Rep 7, 883-97, 2014). Hence, blocking cellular distribution of LE-cholesterol inhibits cell migration and invasion in 2- and 3D environments and improves the ability of Src inhibitors to reduce cell migration. Similarly, upregulation of Annexin A6 (AnxA6), which induces an NPC1-mutant like phenotype, was associated with Stx6 and integrin accumulation in recycling endosomes, leading to reduced cell migration in wound healing, individual cell tracking as well as transwell and 3D-migration/invasion assays (J Biol Chem 291, 1320-35, 2016). Mechanistically, we found that AnxA6 recruits TBC1D15, a Rab7-GTPase activating protein (Rab7-GAP) to cholesterol-rich LE. The ability of AnxA6 to promote Rab7/TBC1D15 assembly downregulates Rab7 GTPase activity and its capacity to promote LE-cholesterol export. Moreover, AnxA6 depletion elevated Rab7-GTP levels and released LE-cholesterol in NPC1 mutant cells. This coincided with increased interaction of Rab7 with ORP1L, a cholesterol-binding and -transfer protein in LE. AnxA6-mediated recruitment of TBC1D15 to LE controls Rab7 activity required for ORP1L-mediated cholesterol transfer out of LE. Loss of AnxA6 and increased Rab7 levels in metastatic prostate cancers indicate that LE-cholesterol transport and AnxA6-dependent Rab7 activity could critically contribute to cancer aggressiveness.