Objective To investigate the feasibility of preparing individualized navigation templates for pedicle screw insertion using a three-dimensional printing forming technique, and to evaluate the accuracy of template-assisted pedicle screw insertion. Methods Five male Chongming goats were selected and total spine CT-scan was performed. After the original Dicom data were imported into the Mimics^® software, the C_3-6, T_3-6 and L_3-6 spinal column models were created to design and generate the cavity models based on a three-dimensional printing forming technique. The navigation template was created with a well-established pedicle screw path using a free-hand procedure, and then bilateral pedicle screw insertion was done assisted by the navigation templates. CT-scan was taken again postoperatively to access the position of the screws. Results Totally 60 navigation templates were prepared and 119 pedicle screws were implanted under the assistance of these templates, with one pedicle screw insertion failed due to a congenital deformity. CT-scan axial images indicated that 114 pedicle screws were completely contained within the pedicles, while 5 screws perforating the pedicle walls. Sagittal images showed accurate screw placement except for 2 screws perforating into the foramen. Conclusion The present study indicates that Mimics^® software is a feasible and precise method for establishing spinal column and related cavity model based on CT-scan images. Pedicle screw insertion assisted by a three-dimensional printing model is highly accurate and easy to perform, which may be a new alternative to conventional technique.