Boots are often used in sports, occupations, and rehabilitation. However, there are few studies on the biomechanical alterations after wearing boots. The current study aimed to compare the effects of running shoes and boots on running biomechanics. Kinematics and ground reaction forces were recorded from 17 healthy males during running at 3.3 m/s with shoe and boot conditions. Temporal distance gait variables, ground reaction force components as well as lower limb joints angle, moment, and power were compared using Paired t-test and Statistical Parametric Mapping package for time-series analysis. Running with boots was associated with greater stride, step, flight, and swing times, greater flight length, and smaller cadence (p<0.05). The only effect of boots on lower limb joints kinematics during running was a reduction in ankle range of motion (p<0.05). Significantly greater hip flexor, abductor, and internal rotator moments, greater knee extensor and abductor moments, and ankle plantar flexor moments were observed at push-off phase of running as well as greater ankle dorsiflexor moment at early-stance in boot condition (p<0.05). Also, knee joint positive power was greater with a significant temporal shift in boot condition, suggesting a compensatory mechanism in response to limited ankle range of motion and the inability of the ankle joint to generate the required power. Our findings showed that running with boots is physically more demanding and is associated with a greater net contribution of muscles spanning hip and knee joints in order to generate more power and compensate for the ankle joint limitations, consequently, may increase the risk of both musculoskeletal injuries and degenerative joint diseases.