Activity-dependent insertion of the tropomyosin-related kinase B (TrkB) receptor into the plasma membrane can explain, in part, the preferential effect of brain-derived neurotrophic factor (BDNF) on active neurons and synapses; however, the underlying molecular mechanisms remain obscure. Here, we report a novel function for carboxypeptidase E (CPE) in controlling chemical long-term potentiation (cLTP) stimuli-induced TrkB surface delivery in hippocampal neurons. Total internal reflection fluorescence (TIRF) assays and line plot assays showed that CPE facilitates TrkB transport from dendritic shafts to the plasma membrane. The Box2 domain in the juxtamembrane region of TrkB and the C-terminus of CPE are critical for the activity-dependent plasma membrane insertion of TrkB. Moreover, TAT-CPE452-466, which could block the association between CPE and TrkB, significantly inhibited neuronal activity-enhanced BDNF signaling and dendritic spine morphological plasticity in cultured hippocampal neurons. Microinfusio...