In order to detect and investigate neuronal firing activity during high-frequency stimulation (HFS), thereby to reveal the mechanisms of deep brain stimulation (DBS) in the treatment of neurological diseases, this project studied the changes of spike waveforms during HFS. Orthodromic-HFS (O-HFS) trains with a duration of 1～2 min and a frequency of 100 or 200 Hz were applied to the afferent fibers (i.e., the Schaffer collaterals) of the hippocampal CA1 region of anesthetized rats. Multi-channel spike signals were recorded in the downstream area of HFS by a microelectrode array. The features of the spike waveforms, such as amplitude and half-height width, were extracted from the sorted unit spikes of interneurons activated by HFS through single synaptic transmissions. The results show that the spike amplitudes decreased and the half-height widths increased significantly during HFS. The percent ratios of the waveform features, compared to their baseline values, show that the amplitude of falling-phase and the amplitude of rising-phase decreased by ～20% and ～40%, respectively, while the half-height widths increased by more than 10%. In addition, the decrease of amplitudes (up to 50%) and the increase of half-height widths (up to 20%) were enhanced during synchronized firings of a large population of neurons or during the O-HFS trains of 100 Hz, a stimulation frequency causing greater excitation to neurons than 200 Hz. Presumably, the excitation of O-HFS could elevate the membrane potentials of downstream neurons and change the kinetics of the ionic channels of the membrane, thereby result in the waveform changes of action potentials. The results support the hypothesis that DBS has excitatory modulation effect on neurons. The study provides guidance for accurately analyzing neuronal unit spike activity during high-frequency stimulation and also provides important clues for revealing the mechanisms underlying the treatment of brain diseases by DBS.