Purpose: The aim of this study was to evaluate the Knoop hardness number (KHN) of dentin and the microtensile bond strength (µTBS) at different walls of MOD preparations, and the correlation between µTBS and the KHN of dentin. Materials and Methods: Human molars with MOD preparations were allocated into 3 groups according to the preparation wall (axial, gingival, pulpal). Each tooth was sectioned into halves, resulting in two similar preparation walls: one was used for measuring the KHN of dentin and the other for µTBS testing. Flat surfaces of the respective walls were obtained by cutting away adjacent walls. For the KHN measurement, twelve indentations were performed along the preparation wall (50 g/15 s). For µTBS testing, specimens were distributed into 3 groups according to the adhesive system (Adper Single Bond Plus/SB, Clearfil SE Bond/SE, Adper Prompt/AP). Composite blocks were built over surfaces; specimens were sectioned (3 slabs/wall) and trimmed to an hourglass shape (1 mm2). Results: The KHN of dentin walls was significantly different (ANOVA/Tukey); pulpal wall > axial wall > gingival wall. The effect of preparation walls on µTBS was dependent on the bonding system (2-way ANOVA/Tukey's test). SB had higher bond strength than the other systems at the gingival wall; and higher than SE at the axial wall. At the pulpal wall, SE presented a higher mean compared to AP. A significant negative correlation between µTBS and KHN was observed at the gingival wall. Conclusion: It could be concluded that dentin structure can affect bond strengths, but the impact of the substrate varies according to the type of adhesive system. Keywords: adhesion, dentin, adhesive systems