Action perception may involve a mirror-matching system, such that observed actions are mapped onto the observer’s own motor representations. The strength of such mirror system activation should depend on an individual’s experience with the observed action. The motor interference effect, where an observed action interferes with a concurrently executed incongruent action, is thought to arise from mirror system activation. However, this view was recently challenged. If motor interference arises from mirror system activation, this effect should be sensitive to prior sensorimotor experience with the observed action. To test this prediction, we measured motor interference in two groups of participants observing the same incongruent movements. One group had received brief visuo-motor practice with the observed incongruent action, but not the other group. Action observation induced a larger motor interference in participants who had practiced the observed action. This result thus supports a mirror system account of motor interference.