Molecular mechanisms underlying long-term neurotrophic regulation of synaptic transmission and plasticity are unknown. We report here that long-term treatment of neuromuscular synapses with glial cell line-derived neurotrophic factor (GDNF) potentiates spontaneous and evoked transmitter release, in ways very similar to presynaptic expression of the Ca2+ binding protein frequenin. GDNF enhances the expression of frequenin in motoneurons, and inhibition of frequenin expression or activity prevents the synaptic action of GDNF. GDNF also facilitates Ca2+ influx into the nerve terminals during evoked transmission by enhancing Ca2+ currents. The effect of GDNF on Ca2+ currents is blocked by inhibition of frequenin expression, occluded by overexpression of frequenin, and is selective to N-type Ca2+ channels. These results identify an important molecular target that mediates the long-term, synaptic action of a neurotrophic factor.