OBJECTIVE Molar-incisor malformation (MIM) is a newly discovered type of dental anomaly that involves a characteristic root malformation of the permanent first molars. The aim of this study was to reveal the microstructure of MIM teeth in order to determine their origin. METHODS Four MIM teeth were extracted from a 9-year-old girl due to severe mobility. The detailed microstructure of the teeth was determined by examinations with micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, immunohistochemical staining, and scanning electron microscopy to reveal the detailed microstructure. RESULTS Micro-CT and H&E staining revealed the pulpal floor comprising three layers: upper, middle, and lower. Amorphous hard tissues and hyperactive cells were observed in the middle layer of the pulpal floor, and the cells stained positively for dentin sialoprotein and osteocalcin, but not for collagen XII. CONCLUSIONS The results of the present study imply that MIM-affected molars probably result from inappropriate differentiation of the apical pulp and dental follicle.