Statement of Problem: Laser irradiation makes structural and chemical changes on the dental hard tissues. These changes alter the level of solubility and permeability of dentin.Purpose: The aim of this study was to compare the microhardness and the structural changes in the dentin cavity floor prepared with Er: YAG laser and bur.Material and Methods: In this experimental study, fifteen intact human molars were selected. Two square cavities were prepared on the buccal and lingual surfaces of each tooth. One side was randomly prepared by Er:YAG laser and the other side by bur. The specimens were divided into two halves. Consequently, there were 30 samples in every group. One half was assigned for the Vickers’s hardness test and the other one, for determination of Ca and P percentage and atomic elements analysis. The data were analyzed by Paired T-tests through SPSS16 (α≤o.o5).Results: The means and the standard deviation of the microhardness were 69.77±25.62 and 51.33±9.31 Kg/mm2 in the laser and bur groups, respectively. Statistical analysis showed significant differences between the two groups (p=0.017). Weight percentage of calcium in the laser cavity (65.5) was less than the bur cavities (68.21) and the difference was significant (p= 0.037).Conclusion: The hardness of dentin in laser group was higher than the bur group because of the higher mineral content of the dentin. The hardness and the mineral content of dentin are important factors in the bonding effectiveness of the dental materials so with laser cavity preparation, good mineral substrate are available for a better bonding.