Background: According to the guideline of the United States center for disease control (CDC), the extracted teeth should be sterilized by autoclaving or storage in 10% formalin before using for educational or research purposes. The objective of this study was to evaluate the effect of this protocol on microhardness of dentin and enamel. Material and Methods: Thirty extracted single-root teeth were used in this study. The crowns were resected, and the roots were longitudinally sectioned into two halves. The Vickers microhardness (VHN) of specimens was measured on polished canal dentin and buccal enamel surfaces. The crowns were randomly divided into three groups (n=10). Group 1 and 2 were sterilized using autoclave and formalin, respectively while group 3 (control) was stored in synthetic tissue fluid. The root halves were also randomly divided into 3 groups (n=20) which were treated as mentioned above for crown samples. Following sterilization, VHN of samples was measured again. ANOVA and paired samples t-tests were used to analyze the data. Results: Autoclaving caused a significant reduction in microhardness of dentin ( P <0.001, 12.04% decreases in VHN). However, there were no significant differences for before and after sterilization within other groups. Conclusions: Based on the results of this study, the CDC protocol is recommended in studies related to enamel microhardness. However, Autoclaving is not an appropriate sterilization method in studies related to dentin microhardness. In these studies, two-week immersion in 10% formalin is recommended.