Purpose: This study aims to investigate the acute effects of shoe midsole stiffness on the joint biomechanics of the lower extremities during specific basketball movements. Methods: Thirty participants wearing stiff midsole shoes (SS) and control shoes (CS) performed layup jumps (LJs) while the kinematics and ground reaction forces were simultaneously collected via the Vicon motion capture system and Kistler force plates. Furthermore, the joint angles, range of motion (ROM), joint power, joint energy, and jump height were calculated. Results: No significant differences were observed between SS and CS conditions for both jump height and the metatarsophalangeal (MTP) joint biomechanics except that the minimum angular velocity of the MTP joint was significantly lower in SS the condition. However, the ROM in the ankle joint was significantly greater in the SS condition than in the CS condition (p < 0.05). Additionally, the maximum plantarflexion power, energy absorption (EA), and energy generation (EG) in the ankle joint were significantly greater in the SS condition than in the CS condition (p < 0.05). Compared with the CS condition, jump height in the SS condition did not increase. Conclusion: During a single LJ, the longitudinal midsole stiffness did not influence the jump height and MTP joint biomechanical patterns but significantly increased the maximum power, EA, and EG during the push-off phase of the ankle joint. These preliminary results indicate that wearing SS could change the ankle joint mechanical patterns by modulating the lower extremity kinetic chain, and may enhance muscle strength in the ankle.