Background: Knowledge of acromioclavicular (AC) joint kinematics and distance may provide insight into the biomechanical function and development of new treatment methods. However, accurate data on in vivo AC kinematics and distance between the clavicle and acromion remain unknown. Purpose/Hypothesis: The purpose of this study was to investigate 3-dimensional AC kinematics and distance during arm elevation in abduction, scaption, and forward flexion in a healthy population. It was hypothesized that AC kinematics and distance would vary with the elevation angle and plane of the arm. Study Design: Controlled laboratory study. Methods: A total of 19 shoulders of healthy participants were enrolled. AC kinematics and distance were investigated with a combined dual fluoroscopic imaging system and computed tomography. Rotation and translation of the AC joint were calculated. The AC distance was measured as the minimum distance between the medial border of the acromion and the articular surface of the distal clavicle (ASDC). The minimum distance point (MDP) ratio was defined as the length between the MDP and the posterior edge of the ASDC divided by the anterior-posterior length of the ASDC. AC kinematics and distance between different elevation planes and angles were compared. Results: Progressive internal rotation, upward rotation, and posterior tilt of the AC joint were observed in all elevation planes. The scapula rotated more upward relative to the clavicle in abduction than in scaption ( P = .002) and flexion ( P = .005). The arm elevation angle significantly affected translation of the AC joint. The acromion translated more laterally and more posteriorly in scaption than in abduction ( P < .001). The AC distance decreased from the initial position to 75° in all planes and was significantly greater in flexion ( P < .001). The MDP ratio significantly increased with the elevation angle ( P < .001). Conclusion: Progressive rotation and significant translation of the AC joint were observed in different elevation planes. The AC distance decreased with the elevation angle from the initial position to 75°. The minimum distance between the ASDC and the medial border of the acromion moved anteriorly as the shoulder elevation angle increased. Clinical Relevance: These results could serve as benchmark data for future studies aiming to improve the surgical treatment of AC joint abnormalities to restore optimal function.