Patients with mechanic ankle instability experience increased tibiotalar and subtalar joint laxity. However, in vivo joint kinematics in functional ankle instability (FAI) patients and lateral ankle sprain (LAS) copers, especially during dynamic activities, are poorly understood. Ten FAI patients, 10 LAS copers, and 10 healthy controls were included in this study. A dual fluoroscopic imaging system was used to analyze the tibiotalar and subtalar joint kinematics during stair descent. Five key poses of stair descent were analyzed. Kinematic data from six degrees of freedom were calculated utilizing a solid modeling software. The range of motion and joint positions in each degree of freedom were compared among the three groups. The tibiotalar joints of FAI patients and LAS copers were significantly more inverted than those of healthy controls during the foot strike (p = 0.016, η p 2 = 0.264). The subtalar joints of FAI patients were significantly more anteriorly translated (pose 2, p = 0.003, η p 2 = 0.352; pose 3, p < 0.001, η p 2 = 0.454; pose 4, p = 0.004, η p 2 = 0.334), inverted (pose 4, p = 0.027, η p 2 = 0.234; pose 5, p = 0.034, η p 2 = 0.221), and externally rotated (pose 4, p = 0.037, η p 2 = 0.217; pose 5; p = 0.004, η p 2 = 0.331) than those of healthy controls during the mid-stance and the heel off. The FAI patients showed excessive tibiotalar inversion and subtalar joint hypermobility during stair descent. Meanwhile, the LAS copers maintained subtalar joint stability, and only showed excessive tibiotalar inversion in foot strike. These data provide insight into the mechanisms behind the development of FAI after initial LAS.