The purpose of this study was to investigate the morphology of pulp chamber floors in mandibular second molars with a C-shaped canal system. Forty-four extracted mandibular second molars with C-shaped roots were collected from a native Chinese population. After the teeth were scanned by the micro-computed tomography (microCT), the pulp chamber floors were reconstructed three-dimensionally using three-dimensional software. The reconstructed images were classified into four types based on the shape of the pulp chamber floor and the location of the dentin fusion between the peninsular-like floor and the pulp chamber wall. Of the 44 reconstructed pulpal floors, 38 (86.37%) were C-shaped (peninsula-like floor) in which 8 (18.18%) had a continuous C-shaped orifice (type I); 16 (36.37%) had a dentin fusion between the peninsula-like floor and buccal pulp chamber wall, forming one or two mesial orifice(s) and a distal orifice (type II); and 14 (31.82%) had a dentin fusion between the peninsula-like floor and the mesial pulp chamber wall, forming a large mesiobuccal-distal (MB-D) orifice and a small mesiolingual orifice (type III). Six (13.63%) teeth did not have a C-shaped floor configuration (type IV). The number and configuration of the canals at different levels below the orifices were analyzed. The results suggested that most teeth in this study with C-shaped roots also have a C-shaped pulpal floor and that a new classification of pulpal floor anatomy would be helpful in locating the C-shaped canals.