Stair ambulation is more physically demanding than level walking because it requires the lower-limb muscles to generate greater net joint moments. Although lower-limb joint kinematics and kinetics during stair ambulation have been extensively studied, relatively little is known about how the lower-limb muscles accelerate the whole-body center of mass (COM) during stair ascent and descent. The aim of the current study was to evaluate differences in muscle contributions to COM accelerations between level walking and stair ambulation in 15 healthy adults. Three-dimensional quantitative gait analysis and musculoskeletal modeling were used to calculate the contributions of the individual lower-limb muscles to the vertical, fore-aft and mediolateral accelerations of the COM (support, progression, and balance, respectively) during level walking, stair ascent and stair descent. Muscles that contribute most significantly to the acceleration of the COM during level walking (hip, knee, and ankle extensors) also dominate during stair ambulation, but with noticeable differences in coordination. In stair ascent, gluteus maximus accelerates the body forward during the first half of stance and soleus accelerates the body backward during the second half of stance, opposite to the functions displayed by these muscles in level walking. In stair descent, vasti generates backward and medial accelerations of the COM during the second half of stance, whereas it contributes minimally during this period in level walking. Gluteus medius performs similarly in controlling mediolateral balance during level walking and stair ambulation. Differences in lower-limb muscular coordination exist between stair ambulation and level walking, and our results have implications for interventions aimed at preventing stair-related falls.