Amyloid precursor protein (APP) is processed sequentially by β-site APP cleaving enzyme (BACE) and γ-secretase to generate amyloid β (Aβ) peptides, one of the hallmarks of Alzheimer's disease. Endosomes or the trans-Golgi network (TGN) are suggested as major subcellular compartments favorable for Aβ production; however, it is still controversial where this process actually occurs. In this study, we investigated the role of different post-endocytic trafficking events in Aβ production using an RNA interference (RNAi) approach. Depletion of Hrs and Tsg101 acting early in the multivesicular bodies (MVB) pathway retained APP in early endosomes and reduced Aβ production. Conversely, depletion of CHMP6 and VPS4 acting late in the pathway rerouted endosomal APP to the TGN for enhanced APP processing. We also showed that VPS35-mediated APP recycling to the TGN was required for efficient Aβ production. Interfering with the bidirectional trafficking of APP between the TGN and endosomes, particularly retromer-mediated retrieval of APP from early endosomes to the TGN, resulted in the accumulation of endocytosed APP in early endosomes with reduced APP processing. These data suggested that Aβ was generated predominantly in the TGN, from the endocytosed pool of APP that had recycled from early endosomes to the TGN.