The relationship between individual muscle dynamics and whole-body metabolic cost is not well established. Here we use <span> to modulate triceps surae (TS) activity during walking to test the following hypotheses based on basic principles of muscle physiology:​ (1) increased TS activity would increase metabolic cost via shorter muscle fascicle lengths and thus reduced force capacity and (2) decreased TS activity would decrease metabolic cost via longer muscle fascicle lengths and thus increased force capacity. 23 young adults walked on an instrumented treadmill at 1.25 m/s using electromyographic (EMG) biofeedback to match targets corresponding to ±20 and ±40% TS activity during push-off (late stance). B-mode ultrasound imaged the medial (MG). Participants increased net metabolic power up to 85% and 21% when targeting increased and decreased TS activity, respectively (p < 0.001). At the instant of peak gastrocnemius force, MG fascicle length was 7% shorter (p < 0.001) and gastrocnemius force was 6% larger (p < 0.001) when targeting + 40% TS activity. Fascicle length was 3% shorter (p = 0.004) and force was 7% lower (p = 0.010) when targeting −40% TS activity;​ participants were unable to achieve decreased activation targets. MG fascicle length and activity mediated 11.7% (p = 0.036) and 57.2% (p = 0.006) of the changes in net metabolic power, respectively. MG force did not mediate changes in net metabolic power (p = 0.948). These findings suggest that changes in the functional operating length of muscle, induced here by volitional changes in TS activity, mediated changes in the metabolic cost of walking, relatively independently of force. Thus, shifts to shorter fascicle lengths (e.g., aging) may mediate activity-induced increases in metabolic cost.</span>