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Background: In endoscopic operations, direct binocular view, tissue sensation and depth perception get lost. It is still unclear whether the novel three-dimensional (3D) high-definition (HD) cameras are able to compensate the limited senses and how this affects the skill set of users with different endoscopic experience. This study aimed first to evaluate if the 3D technology improves depth perception, precision and space orientation as compared to conventional two-dimensional (2D) HD technology. The second aim was to determine the 3D influence on participants with different endoscopic experience. Methods: A total of 24 participants of different experience levels performed three different tasks on a pelvic trainer using the same thoracoscopic unit in 2D and 3D modes. Results were statistically analysed using Student's t-test and Pearson's product–moment correlation. Results: Across all the participants, we found that 3D optic vision significantly reduced the needed time to perform a defined difficult task in comparison to 2D. This difference was less pronounced in participants with higher experience level. Participants with eyeglasses performed slower in both 2D and 3D in comparison to participants with normal vision. Only participants with normal vision could significantly improve their completion times with 3D optic vision. Conclusions: By testing the novel generation of 3D HD cameras, we could demonstrate that the 3D optic of these systems improves depth perception and space orientation for novices and experienced users and especially inexperienced users benefit from 3D optic.