The Latarjet procedure is an established surgical treatment for recurrent glenohumeral joint instability with glenoid bone loss. Intraoperatively, the conjoint tendon is routed through a split in subscapularis to the coracoid bone graft, which is fixed to and augments the anteroinferior glenoid. The objective of this in vitro study was to quantify the influence of glenohumeral joint position and conjoint tendon force on the lines of action and moment arms of subscapularis muscle sub‐regions after Latarjet surgery. Eight fresh‐frozen, entire upper extremities were mounted onto a testing apparatus, and a cable system used to apply physiological muscle loading to the major shoulder muscles. The lines of action and moment arms of four sub‐regions of subscapularis (superior, mid‐superior, mid‐inferior, and inferior) were quantified radiographically with the conjoint tendon unloaded and loaded while the shoulder was in (i) 0° abduction (ii) 90° abduction (iii) 90° abduction and full external rotation (ABER), and (iv) the apprehension position, defined as ABER with 30° horizontal extension. Conjoint tendon loading after Latarjet surgery significantly increased the inferior inclination of the lines of action of the mid‐inferior and inferior sub‐regions of subscapularis in the scapular plane in ABER and apprehension positions (p<0.001), as well as decreased the horizontal flexion moment arm of the inferior subscapularis (p=0.040). Increased subscapularis inferior inclination may ultimately increase inferior joint shear potential, while smaller horizontal flexion leverage may reduce joint flexion capacity. The findings have implications for Latarjet surgical planning and post‐operative rehabilitation prescription.This article is protected by copyright. All rights reserved.