The single leg supine bridge (SLB) is a commonly employed strengthening exercise and is used as a clinical test for hamstring function in sports, however, little is known about the patterns of muscle activation in this task. To explore these activation patterns, nine healthy, recreationally active males underwent functional magnetic resonance imaging (fMRI) of their thighs at rest and immediately after 5 sets of 10 repetitions of the SLB exercise. Exercise-induced increases in the transverse (T2) relaxation time of the biceps femoris long and short heads, semitendinosus and semimembranosus, were determined via signal intensity changes in pre- and post-exercise images and used as an index of muscle activation. The Bonferroni adjusted alpha was set at p<0.008. The semitendinosus exhibited a greater T2 increase than the biceps femoris short head (p<0.001, d=2.0) and semimembranosus (p=0.001, d=1.2), but not biceps femoris long head (p=0.029, d=0.9). Furthermore, the percentage change in T2 for biceps femoris long head was greater than its short head (p=0.003, d=1.4). During the SLB exercise, the semitendinosus is most selectively targeted and the biceps femoris long head is preferentially activated over its short head. These findings may have implications for the use of the SLB in hamstring injury prevention and rehabilitation programs.