Objective: Transfixation of the acromioclavicular (AC) joint is a well-established technique for treating Rockwood IV to VI lesions. However, several complications, including pin breakage or pin migration due to incorrect placement, have been reported in the literature. A cadaveric study was performed to investigate whether the use of 3D navigation might improve the accuracy of AC joint transfixation. Methods: Seventeen transfixations of the AC joint (8 non-navigated, 9 navigated) were performed minimally invasively in cadaveric shoulders. For the navigated procedures, a 3D C-arm (Ziehm Vision FD Vario 3D) and a navigation system (BrainLab VectorVision) were used. Reference markers were attached to the spina scapulae, then a 3D scan was performed and the data transferred to the navigation system. Two Kirschner wires (K-wires) were placed either freehand under fluoroscopic control (in the non-navigated group) or with the use of a navigated drill guide. Radiological analysis was performed with OsiriX software, measuring the distance of the K-wires from the center of the AC joint. For statistical analysis, Student's t-test was performed, with the significance level being set to p < 0.05. Results: The maximum distance of the K-wires from the center of the AC joint was 5.4 AE 1.1 mm for the freehand nonnavigated group and 3.1 AE 1.6 mm for the navigated group (p ¼ 0.0054). The minimum distance of the K-wires from the AC joint center was 3.0 AE 0.6 mm for the freehand group and 1.6 AE 0.6 mm for the navigated group (p ¼ 0.0002). The radiation time was significant lower for the freehand group (41.25 AE 20.4 seconds versus 79.5 AE 13.3 seconds for the navigated group, p ¼ 0.004). There was no statistical difference between the groups with respect to the time required for surgery (11.25 AE 3.6 min for the freehand group and 12.6 AE 4.6 min for the navigated group; p ¼ 0.475). In the freehand group, the AC joint was penetrated by both K-wires in 87.5% of the procedures, compared to 100% in the navigated group. Both K-wires were placed completely intraosseously in the clavicula in 50% of the procedures in the freehand group, compared to 88% in the navigated group. Conclusion: Three-dimensional navigation may improve the accuracy of AC joint transfixation techniques. However, the radiation time is increased when using the navigated procedure, while the overall operation time remains comparable. Nevertheless, a 3D C-arm with a variable isocentric design is recommended for the acquisition of the shoulder scans.