Objective：To investigate the root anatomy and canal configuration of the mandibular first premolars with radicular groove in root surface by micro-computer tomography（Micro-CT）. Methods：In total of 58 mandibular first premolars with radicular groove were selected. The 3-dimensional images of the teeth and canal system morphology were reconstructed by Micro-CT. The types of canal configuration were classified，and the position and angle of Vertucci V root canal bifurcation were measured. The location and incidence of the accessory canals were recorded. The location of the apical foramen，the wall thickness and the diameter of the root canal were analyzed. Results：The most common type of root canal in mandibular first premolar with radicular groove was type V canal. In this study，cases of type V accounted for 55.2%. Root canal furcation of type V mandibular first premolar located about 6.13 mm below the cemento-enamel junction（CEJ）. Significant differences were found in the apical position，wall thickness and root canal diameter between the buccal and lingual canal. Conclusion：The root canal morphology of mandibular first premolars with radicular groove was complex，with a high incidence of type V canals. Understanding the anatomic features of the root canals provides greatly help in clinical treatment.