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Objectives: The aim of this study was to compare the permeability of dentin after using diamond bur and Er:YAG laser. Materials and Methods: Seventy-two recently extracted, intact, and restoration-free human permanent molars were used in this study. The samples were randomly divided into three groups of 24 each and class I cavities were prepared as follows. Group 1: High speed diamond bur with air and water spray. Group 2: Er:YAG laser. Group 3: Er:YAG laser followed by additional sub-ablative laser treatment. Each group consisted of two subgroups with different cavity depths of 2mm and 4mm. The entire cavity floor was in dentin. Two samples from each subgroup were observed under scanning electron microscope (SEM). The external surfaces of other samples were covered with nail varnish (except the prepared cavity) and immersed in 0.5% methylene blue solution for 48 hours. After irrigation of samples with water, they were sectioned in bucco-lingual direction. Then, the samples were evaluated under a stereomicroscope at ×160 magnification. The data were analyzed using two-way ANOVA and Tukey’s HSD test. Results: Two-way ANOVA showed significant difference in permeability between groups 2 and 3 (laser groups with and without further treatment) and group 1 (bur group). The highest permeability was seen in the group 1. There was no significant difference in dentin permeability between groups 2 and 3 and no significant difference was observed between different depths (2mm and 4mm). Conclusion: Cavities prepared by laser have less dentin permeability than cavities prepared by diamond bur.