The reverse fly machine is a popular exercise for strengthening the horizontal shoulder abductors including the posterior deltoid. There seems to be little consensus as to which hand position most effectively targets the posterior deltoid despite this option on most machines. This study investigated the impact of varying one's hand position, and consequently altering shoulder joint rotation, on muscle activity in various glenohumeral muscles during exercise on the reverse fly machine. Nineteen resistance-trained men (mean age = 23.2 ± 4.3 years; height = 176.9 ± 7.1 centimeters; body mass = 81.3 ± 10.5 kilograms; body mass index = 25.9 ± 2.6) were recruited from a university population to participate in the study. In a repeated measures design, subjects grasped the hand bars on the machine with either a pronated (PRO) or neutral (NEU) grip and performed dynamic horizontal abduction repetitions to muscular failure using a load equating to approximately 75% body weight. The order of performance of the hand positions was counterbalanced between participants so that approximately half of the subjects performed PRO first and the other half performed NEU first. Surface electromyography was used to record both mean and peak muscle activity of the posterior deltoid, middle deltoid, and infraspinatus. Results showed that mean electromyography activity for the posterior deltoid was significantly greater in NEU compared with PRO (p = 0.046; 95% CI = 0.1-7.4% maximal voluntary isometric contraction). Similarly, mean electromyography activity of the infraspinatus also was significantly greater in NEU compared with PRO (p = 0.002; 95% CI = 3.7-13.6% maximal voluntary isometric contraction). The results of this study show that performing exercise on the reverse fly machine with a neutral hand position significantly increases activity of the posterior deltoid and infraspinatus muscles compared with a PRO hand position.