Axonal excitation has been proposed as a key mechanism in therapeutic brain stimulation. In this study we examined how high frequency stimulation (HFS) of subcortical white matter tracts projecting to motor cortex affects downstream post-synaptic responses in cortical neurons. Whole-cell recordings were performed in the primary motor cortex (M1) and ventral thalamus of rat brain slices. In M1, neurons showed only an initial depolarization in response to HFS, after which the membrane potential returned to pre-stimulation levels. The prolonged suppression of excitation during stimulation was neither associated with GABAergic inhibition nor complete action potential failure in stimulated axons. Instead we found that HFS caused a depression of excitatory synaptic currents in post-synaptic neurons that was specific to the stimulated subcortical input. These data are consistent with the hypothesis that axonal HFS produces a functional deafferentation of post-synaptic targets likely from depletion of neurotransmitter.