The aim of the present study was to establish the behavior of human medial gastrocnemius (GM) muscle fascicles during stair negotiation. Ten healthy male subjects performed normal stair ascent and descent at their own comfortable speed on a standard-dimension four-step staircase with embedded force platforms in each step. Kinematic, kinetic and electromyographical data of the lower limbs were collected. Real-time ultrasound scanning was used to determine GM muscle fascicle length changes. Musculotendon complex (MTC) length changes were estimated from ankle and knee joint kinematics. The GM muscle was mainly active during the push-off phase in stair ascent and the muscle fascicles contracted nearly isometrically. The GM muscle was mainly active during the touch-down phase of stair descent where the MTC was lengthened, however, the GM muscle fascicles shortened by about 7 mm. These findings show that the behavior and function of GM muscle fascicles in stair negotiation is different from that expected on the basis of length changes of the MTC as derived from joint kinematics.