Background: The external rotation (ER) exercise in performed at a 90° abduction of the shoulder joint is an effective to strengthen the infraspinatus. However, failure of the humeral head to control axial rotation during exercise can be increased the posterior deltoid over activity. Biofeedback training is an effective method of promoting motor learning and control it could look forward to activate the infraspinatus selectively by controlling the humeral head during exercise. Objects: The aim of this study was investigated that whether biofeedback for axial rotation was effective to activate selectively the infraspinatus during ER exercise. Methods: The 15 healthy males participated, and all subjects performed both ER exercise in a sitting position with shoulder abducted 90° under conditions with and without axial rotation biofeedback. Exercise was performed in a range of 90° ER, divided into three phases: concentric, isometric, and eccentric. The infraspinatus and posterior deltoid muscle activity were observed using surface electromyography. Results: Both infraspinatus activity (p < 0.01) and infraspinatus to posterior deltoid activity ratio (p = 0.01) were significantly higher with biofeedback however, posterior deltoid activity was significantly lower with biofeedback (p = 0.01). The infraspinatus muscle activity and muscle activity ratio were the highest in the isometric contraction type, and there were significant differences for all contraction types (p < 0.05). Whereas, the posterior deltoid activity was the lowest in the isometric contraction type, and showed a significant difference between isometric and other two contraction types (p < 0.05), but no significant different between concentric and eccentric contraction. Conclusion: Our results indicate that the axial rotation biofeedback during sitting ER exercise might be effective method to activating selective infraspinatus muscle and recommended to enhance the dynamic stability of the shoulder joint.