Objective: To investigate the differences in dentin structure, mineral content and microhardness of single premolar roots with or without an isthmus. Methods : Thirty single-root premolars with a canal isthmus (experimental group) and thirty single-root premolars without a canal isthmus (control group) were collected and analyzed by scanning electron microscopy, X-ray energy spectrometry and a Vickers microhardness tester. Structure, mineral content and microhardness were compared. Results In the experimental group, the dentinal tubules were not obvious, and the structure was disordered. In the control group, the dentin tubules of the root canal wall were arranged regularly, and the lumen opening was clear. The Ca and P contents and Ca/P ratio of the experimental group were lower than those of the control group (P < 0.05). The microhardness of the experimental group was less than that of the control group (P < 0.05). Results: In the experimental group, the dentinal tubules were not obvious, and the structure was disordered. In the control group, the dentin tubules of the root canal wall were arranged regularly, and the lumen opening was clear. The Ca and P contents and Ca/P ratio of the experimental group were lower than those of the control group (P < 0.05). The microhardness of the experimental group was less than that of the control group (P < 0.05). Conclusion The dentin structure of the root canal isthmus was changed, and the Ca, P contents and microhardness were low. The root canal isthmus was a weak link in the root, which could weaken the resistance of the hard tissue of the root tooth, which may be one of the important factors that could cause vertical root fracture.