Background: Gender has been considered as an influencing factor in the incidence of sports injuries. But few studies have discussed whether gender differences change after long-distance running. This study aimed at investigating whether the kinematic and ground reaction forces (GRFs) differences between males and females were altered by a 5 km run. Methods: Thirty novice runners (15 males and 15 females) with heel strikes were recruited for this study. In the test before and after the 5 km run, the participants were asked to run through the force plate with their right foot at a speed of 3.3 m/s ± 5%. Kinematics data and GRFs were collected synchronously. Each participant completed five successful running trials for further analysis of data. Results: Gender differences existed in ankle sagittal peak angle (pre: p = 0.059; post: p = 0.013), knee frontal peak angle (pre: p = 0.345; post: p = 0.014), knee horizontal nadir angle (pre: p = 0.056; post: p = 0.005), hip frontal nadir angle (pre: p = 0.103; post: p = 0.001) and peak lateral force (pre: p = 0.564; post: p = 0.001) after a 5 km run, but there were no gender differences before a 5 km run. Gender differences in the knee and hip movement in the frontal plane and horizontal plane and anterior-posterior GRFs changed obviously in the stance phase before and after a 5 km run. Conclusions: The gender difference in lower limb biomechanics during running is not constant. Differences change in peak angle, peak lateral force, knee and hip movement in the frontal plane and horizontal plane, and anterior-posterior GRFs between males and females were associated with the different incidence of running-related injuries, such as patellofemoral joint injuries, anterior cruciate ligament injury and iliotibial band syndrome, etc. These changes can give some concrete details to explore the different incidence rates of lower limb sports injuries between genders.