In the dynamic interaction between the runner and the ground, the running shoe is the only medium that bears the impact force of body weight and plays a crucial role in athletic performance. Traditional designs do not adequately consider the different shapes of the foot, which often leads to discomfort and aggravation of foot disorders. This study presents an innovative approach to running shoe midsole design using 3D-printed chiral negative poisson’s ratio (NPR) structures to enhance shock absorption and support, thereby optimizing biomechanical performance and comfort. Using computer-aided design (CAD) and computer-aided engineering (CAE), the biomechanical effects of different midsole structures has been explored through finite element analysis (FEA). The study focuses on optimizing the cushioning, propulsion and stability of the midsole to mitigate the impact on the ankle and knee. Static compression and dynamic impact simulations were utilized to comprehensively select optimized design of midsole structure and the selected structures was 3D printed to validate the biomechanical benefits in a wear trial. The results of the study highlight the superior performance of chiral NPR structures in reducing forefoot stress during standing and movement and advance the design and functionality of 3D printed materials in running shoes.