Studies revealed that navigation systems that provided intraoperative assistance might improve pedicle screw insertion accuracy, and also implied that different systems provided different pedicle screw insertion accuracy. A systematic review and meta-analysis was conducted to focus on the pedicle screw insertion accuracy with or without the assistance of image-guided system, and the variance among the different navigation systems. Comparative studies were searched on pedicle screw insertion accuracy between conventional and navigated method, and among different navigation systems. A total of 43 papers, including 28 clinical, 14 cadaveric and 1 model studies, were included in the current study. For clinical articles, there were 3 randomized clinical trials, 4 prospective comparative studies and 21 retrospective comparative studies. The incidence of pedicle violation among computer tomography-based navigation method group was statistically significantly less than that observed among the conventional group (OR 95% CI, in vivo: 0.32-0.60; in vitro: 0.24-0.75 P < 0.01). Two-dimensional fluoroscopy-based navigation system (OR 95% CI, in vivo: 0.27-0.48; in vitro: 0.43-0.88 P < 0.01) and three-dimension fluoroscopy-based navigation system (OR 95% CI, in vivo: 0.09-0.38; in vitro: 0.09-0.36 P < 0.01) also obtained significant reduced screw deviation rate over traditional methods. Between navigated approaches, statistically insignificant individual and pooled RR values were observed for all in vivo subgroups. Pooled estimate of in vitro studies show that computer tomography-based and three-dimension fluoroscopy-based navigation system provided more accurate pedicle screw insertion over two-dimension fluoroscopy-based navigation system. Our review showed that navigation provided a higher accuracy in the placement of pedicle screws compared with conventional methods. The superiority of navigation systems was obvious when they were applied to abnormal spinal structure. Although no strong in vivo evidence has detected significantly different pedicle screw placement accuracy among the three major navigation systems, meta-analysis revealed the variance in pedicle screw insertion accuracy with different navigation methods.