The effects of a 12-week gait retraining program on the adaptation of the medial gastrocnemius (MG) and muscle-tendon unit (MTU) were investigated. 26 runners with a rearfoot strike pattern (RFS) were randomly assigned to one of two groups: gait retraining (GR) or control group (CON). MG ultrasound images, marker positions, and ground reaction forces (GRF) were collected twice during 9 km/h of treadmill running before and after the intervention. Ankle kinetics and the MG and MTU behavior and dynamics were quantified. Runners in the GR performed gradual 12-week gait retraining transitioning to a forefoot strike pattern. After 12-week, (1) ten participants in each group completed the training; eight participants in GR transitioned to non-RFS with reduced foot strike angles; (2) MG fascicle contraction length and velocity significantly decreased after the intervention for both groups, whereas MG forces increased after intervention for both groups; (3) significant increases in MTU stretching length for GR and peak MTU recoiling velocity for both groups were observed after the intervention, respectively; (4) no significant difference was found for all parameters of the series elastic element. Gait retraining might potentially influence the MG to operate at lower fascicle contraction lengths and velocities and produce greater peak forces. The gait retraining had no effect on SEE behavior and dynamics but did impact MTU, suggesting that the training was insufficient to induce mechanical loading changes on SEE behavior and dynamics.