In biomedical research it is difficult to perceive tumors or cells and perform biopsies manually. Robotics technology can offer a reliable solution for accurate needle insertion. A novel 5 degrees of freedom (DOF) robot for inserting needles into small animal subjects was developed. The robot can realize dexterous alignment of the needle using two parallel mechanisms, and has a syringe mechanism to insert needles to subjects. Operations on small animals require high accuracy positioning during needle insertion. In this paper, kinematic calibration of the 5 DOF robot using an optical tracker as an external sensor is performed to enhance accuracy of the system.