Given the importance of sterilizing human teeth before using them in research, the effects of a 25 kGy dose of gamma radiation on the mechanical properties of enamel and its resistance to demineralization were evaluated. Thirty human teeth were sectioned longitudinally, and while one half of each tooth was irradiated, the other half was kept as a control. Abraded and unabraded enamel slabs were obtained from these halves. The surface microhardness (SMH) of abraded slabs of irradiated and non-irradiated enamel was determined to evaluate the effect of radiation on enamel structure. Further, both abraded and unabraded slabs, either irradiated or non-irradiated, were submitted to a pH-cycling model to evaluate enamel resistance to demineralization, which was quantified by mineral loss (deltaZ) using cross-sectional microhardness. The data for SMH and deltaZ were statistically analyzed by t-test and ANOVA, respectively. The difference in enamel SMH between slabs from irradiated teeth and non-irradiated teeth was not statistically significant (P > 0.05). The abraded enamel slabs showed higher values of deltaZ than unabraded enamel slabs (P < 0.05), but the irradiation effect was not statistically significant (P > 0.05). The results suggest that the medical gamma radiation dose of 25 kGy does not affect either enamel hardness or its resistance to demineralization.