Background Full-can exercise was associated with a significantly higher scapulohumeral rhythm than empty-can exercise. These findings suggest that the movements of the scapula and glenohumeral joint are affected by the position of the arm. Purpose We investigated the effect of arm position on the muscle activity of scapular upward rotators during prone arm lift exercise. Study design Cross-sectional study Methods Thirty-two healthy subjects were participated in this study. The muscle activity of the serratus anterior (SA), upper trapezius (UT), lower trapezius (LT), and infraspinatus (IS) were measured using a surface electromyography device. All subjects were asked to perform prone arm lift exercise according to the three arm positions: palm down (PD), neutral (NT), and palm up (PU). One-way repeated analysis of variance was conducted. Results The EMG activities of the UT, LT, and SA were significantly different among three conditions (PU vs. NT vs. PD; <italic>p</italic><0.05). The muscle activities of the UT and LT were significantly higher in the PD than in the PU (<italic>p</italic><0.017). The muscle activity of SA was significantly higher in the PU than in the PD (<italic>p</italic><0.017). However, the activity of the IS muscle was not significantly different among the three conditions (<italic>p</italic>>0.05). Conclusions Prone arm lift exercise for SA strengthening is recommended to be performed in a PU position.