Brain-derived neurotrophic factor (BDNF) has been implicated in higher-order cognitive functions and in psychiatric disorders such as depression and schizophrenia. BDNF modulates synaptic transmission and plasticity primarily through the TrkB receptor, but the molecules involved in BDNF-mediated synaptic modulation are largely unknown. Myosin VI (Myo6) is a minus end-directed actin-based motor found in neurons that express Trk receptors. Here we report that Myo6 and a Myo6-binding protein, GIPC1, form a complex that can engage TrkB. Myo6 and GIPC1 were necessary for BDNF-TrkB-mediated facilitation of long-term potentiation in postnatal day 12-13 (P12-13) hippocampus. Moreover, BDNF-mediated enhancement of glutamate release from presynaptic terminals depended not only upon TrkB but also upon Myo6 and GIPC1. Similar defects in basal synaptic transmission as well as presynaptic properties were observed in Myo6 and GIPC1 mutant mice. Together, these results define an important role for the Myo6-GIPC1 motor complex in presynaptic function and in BDNF-TrkB-mediated synaptic plasticity.