Objectives <p>This study investigated the nanomechanical properties, microstructure, and composition of dentinogenesis imperfecta type II (DGI-II) peritubular dentin (PTD) and intertubular dentin (ITD) and examined the correlations between them.</p> Materials and methods <p>Six samples from each of the normal and DGI-II groups were prepared by cutting the midcoronal dentin perpendicular to the dentin tubules. The number and morphology of the dentin tubules were then observed by scanning electron microscopy (SEM). Hydroxyapatite (HAP) was detected using high-resolution atomic force microscopy (HR-AFM). The chemical composition was determined using atomic force microscopy-infrared spectroscopy (AFM-IR). The nanomechanical properties were evaluated using amplitude modulation-frequency modulation (AM-FM) techniques. Finally, a multiple linear regression (MLR) model was used to verify the correlations between PTD and ITD.</p> Results <p>SEM of the DGI-II dentin revealed a considerable reduction in the number and area of the tubules. HR-AFM revealed dramatic increases in the HAP particle size and DGI-II dentin nanoscale roughness, especially PTDs. AFM-IR revealed that in the DGI-II groups, the phosphate content decreased in both the PTDs and ITDs, whereas the amide I (A-I) and amide II (A-II) content was elevated in the ITDs. AM-FM testing revealed a considerable reduction in the Young’s modulus and increases in the PTD and ITD indentations in the DGI-II dentin. MLR demonstrated that the changes in microstructure and composition were related to a decrease in the nanomechanical properties of the DGI-II dentin.</p> Conclusions <p>The DGI-II dentin nanomechanical properties deteriorated considerably, especially those of the PTDs, presumably because of alterations in the HAP and chemical composition.</p> Clinical relevance <p>Understanding the nanomechanical properties, microstructure, and composition of DGI-II dentin could help dentists develop novel individualized restorative techniques.</p>