Background: The purpose of this study was to examine whether surgeon handedness could affect cup positioning in manual total hip arthroplasty (THA), and whether robot could diminish or eliminate the impact of surgeon handedness on cup positioning in robot-assisted THA. Methods: Fifty-three patients who underwent bilateral robot-assisted THA and sixty-two patients who underwent bilateral manual THA between August 2018 and July 2019 in our institute were respectively analyzed in this study. When the difference between the bilateral anteversion and inclination was greater than 5°, the patient was regarded as having different cup positioning between bilateral THA. Their demographics, orientation of acetabular cup, and postoperative 3 month Harris hip score (HHS) were recorded for analysis. Results: There were no significant differences in the gender, age, BMI, diagnosis's composition, and preoperative and postoperative HHS between the robotic and manual group. Two left hips dislocated in the manual group. The anteversion of left hip was significantly larger than that of right hip (24.77 ± 10.44 vs 22.44 ± 8.67, p = 0.043) in the manual group. There were no significant differences of cup positioning between bilateral robot-assisted THA. The patients in manual group were significantly more likely to have different cup positioning between bilateral hips than those in robotic group (77% vs 45%, p = 0.000). More manual THA were located out of the target zone than robot-assisted THA (70% vs 48%, p = 0.001). Conclusions: Surgeon's handedness showed a trend towards an impact on cup positioning in manual THA and robot might help surgeon eliminate the adverse impact. However, the impact of handedness on the clinical outcomes still needs further observation.