Inclined walking is a challenging task that requires active neuromuscular control to maintain stability. However, the adaptive strategies that preserve stability during inclined walking are not well understood. Investigating the effects of self-paced inclined treadmill walking on gait stability characteristics and the activation patterns of key lower limb muscles can provide insights into these strategies. The aim of this study was to investigate the effects of self-paced inclined treadmill walking on gait stability characteristics and the activation of key lower limb muscles. Twenty-eight able-bodied individuals (mean age 25.02, SD 2.06 years) walked on an augmented instrumented treadmill for 3 minutes at 3 inclination angles (-8°, 0°, and 8°) at their preferred walking speed. Changes in gait characteristics (ie, stability, walking speed, spatial-temporal, kinematic, and muscle forces) across inclination angles were assessed using a repeated measures ANOVA and the Friedman test. The study revealed that inclined treadmill walking has a significant impact on gait characteristics (P<.001). Changes were observed in spatial-temporal parameters, joint angles, and muscle activations depending on the treadmill inclination. Specifically, stability and walking speed decreased significantly during uphill walking, indicating that it was the most challenging walking condition. Uphill walking also led to a decrease in spatial parameters by at least 13.53% and a 5.26% to 10.96% increase in temporal parameters. Furthermore, joint kinematics and peak activation of several muscles, including the hamstrings (biceps femoris, long head=109.5%, biceps femoris, short head=53.3%, semimembranosus=98.9%, semitendinosus=90.9%), gastrocnemius (medial gastrocnemius=40.6%, lateral gastrocnemius=35.3%), and vastii muscles (vastus intermedius=12.8%, vastus lateralis=16.7%) increased significantly during uphill walking. In contrast, downhill walking resulted in bilateral reductions in spatial-temporal gait parameters, with knee flexion increasing and hip flexion and ankle dorsiflexion decreasing. The peak activation of antagonist muscles, such as the quadriceps, tibialis anterior, and tibialis posterior, significantly increased during downhill walking (rectus femoris=97.7%, vastus lateralis =70.6%, vastus intermedius=68.7%, tibialis anterior=72%, tibialis posterior=107.1%). Our findings demonstrate that able-bodied individuals adopt specific walking patterns during inclined treadmill walking to maintain a comfortable and safe walking performance. The results suggest that inclined treadmill walking has the potential to serve as a functional assessment and rehabilitation tool for gait stability by targeting muscle training. Future research should investigate the effects of inclined treadmill walking on individuals with gait impairments and the potential benefits of targeted muscle training. A better understanding of the adaptive strategies used during inclined walking may lead to the development of more effective rehabilitation interventions for individuals with lower limb injuries.