<h3>Research Objectives</h3> Rotator cuff muscles are commonly regarded as the main stabilizers of the glenohumeral joint, but recent modeling work from our group suggests that muscles which primarily move the shoulder also contribute substantially to glenohumeral stiffness. The objective of this study was to evaluate this possibility in experiments designed to assess the relationship between muscle activity in the primary shoulder movers and glenohumeral stiffness. <h3>Design</h3> Cross-sectional study. <h3>Setting</h3> Biomechanics research laboratory. <h3>Participants</h3> 15 healthy adults (8F/7M, 26±4 years) with no shoulder pain or injury. <h3>Interventions</h3> N/A. <h3>Main Outcome Measures</h3> We measured translational glenohumeral stiffness (resistance to humeral head translation to quantify glenohumeral stability) using a linear motor that applied 15 mm stochastic anterior-posterior perturbations to the proximal humerus. Measurements were made as participants generated submaximal isometric torques. We used system identification to estimate stiffness from the applied forces and measured displacements. Active stiffness was defined as the increase in glenohumeral stiffness during torque generation compared to during no torque generation. Electromyography measurements (EMGs) were recorded from 3 rotator cuff muscles (intramuscular electrodes: supraspinatus, infraspinatus, subscapularis) and 6 primary shoulder movers (surface electrodes: anterior/middle/posterior deltoid, pectoralis major, latissimus dorsi, teres major). We used linear mixed effects models to characterize the relationship between EMGs and active stiffness. <h3>Results</h3> EMGs in rotator cuff muscles and primary shoulder movers were positively correlated with active stiffness. EMGs recorded from only the primary shoulder movers better predicted stiffness during active contractions than EMGs recorded from only the rotator cuff muscles (R2=0.80 vs. R2=0.56; P< 0.001). Including EMGs recorded from all muscles improved the fit compared to the model with only primary shoulder movers (R2=0.84 vs. R2=0.80; P< 0.001), but this difference was small. <h3>Conclusions</h3> Our results provide additional evidence that the primary shoulder movers contribute to glenohumeral stability. The stabilizing potential of these muscles should be considered when designing and implementing rehabilitation protocols used to treat individuals with reduced glenohumeral stability, such as following shoulder dislocations. <h3>Author(s) Disclosures</h3> Funded by the NIH (F31AR074288, T32GM008152, UL1TR001422), the American Society of Biomechanics, and Northwestern University.