Dens invaginatus (D.I.) is a developmental anomaly caused by the infolding of the surface of a tooth crown before calcification has occurred. Its aetiology is controversial and remains unclear. It occurs in all dentitions with a prevalence that ranges from 0.25% to 7.74% and is mostly seen in the maxillary permanent incisors, particularly in the lateral incisors. Posterior teeth are infrequently involved. The purpose of this study was to investigate the morpho-structure of a second upper molar dens invaginatus compared with a control tooth. Ground and decalcified sections were prepared and histo-morphological evaluation of dental tissues was performed by using light microscopy, microradiography, and confocal laser scanning microscopy analysis (CLSM). The mechanical behaviour was tested by means of microhardness (HV) test. The results of our investigation showed structural anomalies of hard tissues, such as a difference in enamel prism diameter, in number and diameter of peripulpal dentinal tubules and in surface and diameter of cementocyte lacunae between D.I. and control tooth.