The insular cortex (IC) processes various kinds of sensory and emotional information. Multiple tooth-loss induces impairment of oral sensory and motor functions, which might result in the up- or down-regulation of signal processing in the IC. In the present study, we investigated how multiple tooth-loss affects neural activities in the IC. Slices of the IC were prepared from control (untreated) rats and rats raised following the loss of their upper molar teeth, and optical recordings with voltage-sensitive dye were made. Electrical stimulation was delivered to the agranular IC (AIC). The velocity of optical signal from the AIC to the granular IC (GIC) decreased in multiple tooth-loss rats compared with control rats. Field potentials from the GIC were recorded. Onset times of evoked response at the GIC recorded from multiple tooth-loss rats were prolonged compared with those recorded from control rats, suggesting that signal velocity in multiple tooth-loss rats had decreased. A reduced signal velocity was accompanied by neuronal loss in the GIC, which was confirmed by counting the cell numbers on Nissl-stained sections. Thus, multiple tooth-loss may have influences on the GIC where signal processing speed decreases.