Introduction: Gamma radiation is usually used, as a primary or supplementary treatment, for oral cancer patients. Aim: The present work was undertaken to investigate, in vitro, the effect gamma irradiation of a cumulative dose of 60 Gy (20 Gy/day over three days) on enamel and dentin microhardness before and after demineralization and remineralization. Materials and methods: Ten healthy freshly extracted human third molars were used in this study. The teeth specimens were cut perpendicular to their occlusal surface into two halves. Half of the specimens were gamma-irradiated to a dose of 60 Gy (20 Gy/day). The data involved the measurement of VHN Vickers microhardness on both radiated and non-irradiated. Results: The results indicated that the mean microhardness values of the non-irradiated enamel samples were decreased after gamma irradiation, and were significantly decreased after demineralization; meanwhile, the microhardness values of the demineralized non-irradiated enamel samples were significantly increased after remineralization. The results showed that the microhardness of the demineralized non-irradiated dentin samples was increased, and that the microhardness of the gamma-irradiated dentin samples was slightly decreased. Conclusions: gamma-irradiation had significant effect on enamel and dentin microhardness values and had significant decrease effect on demineralization of enamel while had significant increase by remineralization.