Background: The majority of research on barefoot running focuses on acute changes in altering footwear without regard to the runner's experience with barefoot or minimalist footwear running. Objective: The objective of this study was to compare the effect of footwear (standard cushioned running shoes, barefoot) and running surface (flat surface, uneven terrain) on gait in experienced runners using minimalist shoes. Methods: Terrain running was simulated by three custom-made mats with randomly placed firmly attached stones. Seven experienced trail runners participated in this study. All participants were forefoot strikers. Participants underwent three-dimensional kinematic and kinetic analysis consisting of five running trials in each combination of footwear and surface. A trial was successful when the participant maintained a velocity of 3.2 ± 0.16 m/s without targeting a force platform. Results: Uneven terrain conditions along with barefoot conditions led to significantly decreased peak moment of ankle plantarflexion (terrain: p = .041, footwear: p = .026) and decreased second peak of vertical ground reaction force in comparison with other conditions (terrain: p = .026, footwear: p = .004). Uneven terrain conditions also significantly decreased ankle dorsiflexion at initial contact with the ground for both footwear conditions (p = .021). Conclusions: We conjecture that net ankle moments could be decreased by barefoot running in terrain conditions in skilled forefoot runners. Experienced runners using minimalist shoes may incorporate trail running into their barefoot running regime without risk of higher Achilles tendon loading compared to even running.