The purpose of this study was to investigate the anatomical features of C-shaped root canal system in mandibular second molars using micro-computed tomography (microCT). Fifty-eight extracted mandibular second molars with fused roots were collected from a native Chinese population. The teeth were scanned into layers of 0.5-mm thickness by microCT and measurements were made at eleven levels. The ratio of the depth of the deepest part of the groove to the buccal-lingual thickness of the cross-section of the root was calculated for each tooth. The canal shapes of the scanned cross-sections were assessed and classified according to a modified Melton's method. Results were subject to the Kruskal-Wallis test. Of the 58 molars, 54 had a C-shaped canal system with a mean groove-to-thickness ratio of 47.96%; the four teeth without a C-shaped canal had a mean ratio of 14.82%. Most orifices (98.1%) were found within 3 mm below the cementoenamel junction. Of teeth with a C-shape canal system, a majority demonstrated an orifice with an uninterrupted "C" configuration. Seventeen canals divided in the apical portion, most of which did so within 2 mm from the apex. The cross-sectional shape varied drastically along the length of the canal. Teeth with a high groove-to-thickness ratio had at least one section with C1, C2, or C3 configuration. The canal shape in middle and apical thirds of C-shaped canal systems could not be predicted on the basis of the shape at the orifice level. Section 2 of this paper addressed the correlation between the radiographic appearance and these microCT images.