Unraveling the quantitative properties of the spatio-temporal cortical responses to microstimulation is of essential importance for designing intracortical neural prostheses. Despite the millisecond-order precision of neuronal spiking and synaptic signaling in the cortex, the circuit response dynamics at such temporal resolution has been investigated to a limited extent. In the present, cortical neural responses to repetitive microstimulation pulses were examined in the mouse cerebral slices by means of the voltage-sensitive dye imaging. The amplitude of the population spikes induced directly by the stimulus pulses successively decreased when the inter-pulse interval of the stimuli was 5 msec, but not 20 msec. This was similar between the experiments in that the stimulation was applied to either the layer II/III or IV. These results suggested that the refractory period of spiking at the population level is one of limiting conditions for designing efficient frequency for repetitive pulse stimulation.