Introduction: Restoration of native head-shaft angle (HSA) is critical for treatment of proximal humerus fracture. However, HSA has not been properly investigated according to the humeral rotation. This study was designed to analyze the relationship between the humeral rotation and the HSA at 1 increments, and clarify its serial changing pattern according to the humeral rotation. Hypothesis: The angulation of HSA would be undervalued when the humerus is being rotated externally and it would be overvalued when it is being rotated internally. Materials and methods: Eight dried cadaveric normal humeri were CT scanned. They were analyzed using computer-aided design with a standardized neutral position. HSA was the angle between the humeral shaft axis (SA) and the humeral head axis (HA). SA and HA were the best-fit lines through center of all the best-fitting circles in every cross section along the humeral shaft and within the humeral head, respectively. Each 3D model was rotated 30 degrees internally and 45 degrees externally relatives to the SA at 1 degrees increments with the camera was fixed at antero-posterior view of neutral position. Angulation of HSA in every rotational degree was documented as ratio relatives to the angulation of HSA in neutral position. Results: The average HSA at neutral position was 133 +/- 1.93 degrees HSA was underestimated by 8 +/- 1.9% and it was overestimated by 20 +/- 5.1% at the maximum external rotation (ER) and internal rotation (IR), respectively. HSA was underestimated by 1% in every 5.8 degrees of ER and overestimated by 1% in every 1.5 degrees of IR. Rotational misalignments within 10 degrees of IR and 18 degrees of ER could be tolerated (P >.05). Conclusions: HSA was underestimated at ER and was overestimated at IR. This information could be useful for surgeons in restoring the native HSA for treatment of proximal humerus fracture.