Gait on inclined surfaces requires active neuromuscular control to maintain stability. This study investigated the effects of self-paced incline treadmill walking on gait stability characteristics and the activation of key lower limb muscles. Twenty-seven healthy subjects (mean age: 25.02 ± 2.06 years) walked at their preferred walking speed on an augmented instrumented treadmill for three minutes at three inclination angles (-8°,0°, and 8°). Changes in gait characteristics (i.e., stability, walking speed, spatial-temporal, kinematic, and muscle forces) across inclination angles were assessed using repeated measures ANOVA and Freidman tests. Results showed that inclined treadmill walking had a significant impact on overall gait characteristics, reflecting changes in gait parameters and muscle activations with respect to the inclination of the treadmill. Stability and walking speed had reduced significantly during uphill walking, suggesting that it was the most challenging walking condition. During uphill walking, there was a significant increase in the peak activation of hamstrings, gastrocnemius, vastus intermedius, and vastus lateralis muscles. In contrast, the peak activation of the antagonist muscle groups including the quadriceps, tibialis anterior, and tibialis posterior muscles significantly increased during downhill walking. Our findings demonstrate that able-bodied individuals adopted walking patterns during inclined treadmill walking to maintain a comfortable and safe walking performance. Future studies should consider inclined treadmill walking as a functional assessment tool or as a rehabilitation intervention to improve gait stability by targeting muscle training.