Action observation (AO), observing another individual perform an action, has been implicated in several higher cognitive processes including forming basic motor memories. Previous work has shown that physical practice (PP) results in cortical motor representational changes, referred to as use-dependent plasticity (UDP), and that AO combined with PP potentiates UDP in both healthy adults and stroke patients. In humans, AO results in activation of the ventral premotor cortex (PMv), however, whether this PMv activation has a functional contribution to UDP is not known. Here, we studied the effects disruption of PMv has on UDP when subjects performed PP combined with AO (PP + AO). Subjects participated in two randomized crossover sessions measuring the amount of UDP resulting from PP + AO while receiving disruptive (1 Hz) TMS over the fMRI-activated PMv or over frontal cortex (Sham). We found that, unlike the sham session, disruptive TMS over PMv reduced the beneficial contribution of AO to UDP. To ensure that disruption of PMv was specifically interfering with the contribution of AO and not PP, subjects completed two more control sessions where they performed only PP while receiving disruptive TMS over PMv or frontal cortex. We found that the magnitude of UDP for both control sessions was similar to PP + AO with TMS over PMv. These findings suggest that the fMRI activation found in PMv during AO studies is functionally relevant to task performance, at least for the beneficial effects that AO exerts over motor training.