Brain-derived neurotrophic factor (BDNF) is essential for neuronal growth, differentiation, and synaptic plasticity. Although the release and effects of BDNF have been well-studied separately, the transfer of BDNF between cells has not been investigated. Using a four-fluorophore imaging approach to identify both the cell of origin and target cells, we quantified the transfer of BDNF. Surprisingly, we found that astrocytes are the main recipient of neuronally expressed BDNF. We further found that astrocytes specifically take up mature (not pro) BDNF released by neurons. Over-expression of TrkB in neurons redirects released BDNF from astrocytes to neurons, indicating that TrkB levels determine neuronal versus astrocytic BDNF uptake. Increased neuronal activity further increased astrocytic (but not neuronal) uptake of neuronally expressed BDNF. Finally, we demonstrate that astrocytes are not simply a sink for excess BDNF, but that BDNF taken up by astrocytes mediates physiological effects on the astrocytic population by increasing astrocytic territory.