Stretching is commonly used prior to exercise, as it is thought to reduce the risk of injury, and it is also used in the preconditioning of tendon grafts. As tendon properties have been shown to be different between genders, it is proposed that stretching will differentially affect the structure. Here we examine the effect of acute stretch on the mechanical properties of both male and female medial gastrocnemius tendon. Female [20 years +/- 1 (SEM), n = 17] and male (22 years +/- 1, n = 18) subjects underwent a 5-min passive dorsiflexion stretch. Prior to and post stretch medial gastrocnemius tendon stiffness (K), length (l) and cross-sectional area (csa) were measured using ultrasonography and dynamometry. Stiffness and Young's modulus (epsilon) were significantly reduced with stretch for both genders (p < 0.05). Females showed significantly (p < 0.05) greater pre- to poststretch decreases in K (22.4 vs. 8.8%) and epsilon (20.5 vs. 8.4%) in comparison to males. The present results show that stretching acutely reduces stiffness of the medial gastrocnemius tendon in females and males, with females showing significantly greater change. The observed disparity between genders may be due in part to variations in tendon moment arm and intrinsic differences in tendon composition. These differential changes in tendon mechanical properties have functional, motor control, and injury risk implications, as well as possible implications for preconditioning of tendon grafts.