BACKGROUND Motor imagery (MI) is the mental performance of movement without muscle activity. It is generally accepted that MI and motor performance have similar physiological mechanisms. OBJECTIVE To investigate the activity and excitability of cortical motor areas during MI in subjects who were previously trained with an MI-based brain-computer interface (BCI). METHODS Eleven healthy volunteers without neurological impairments (mean age, 36 years; range: 24-68 years) were either trained with an MI-based BCI (BCI-trained, n = 5) or received no BCI training (n = 6, controls). Subjects imagined grasping in a blocked paradigm task with alternating rest and task periods. For evaluating the activity and excitability of cortical motor areas we used functional MRI and navigated transcranial magnetic stimulation (nTMS). RESULTS fMRI revealed activation in Brodmann areas 3 and 6, the cerebellum, and the thalamus during MI in all subjects. The primary motor cortex was activated only in BCI-trained subjects. The associative zones of activation were larger in non-trained subjects. During MI, motor evoked potentials recorded from two of the three targeted muscles were significantly higher only in BCI-trained subjects. The motor threshold decreased (median = 17%) during MI, which was also observed only in BCI-trained subjects. CONCLUSIONS Previous BCI training increased motor cortex excitability during MI. These data may help to improve BCI applications, including rehabilitation of patients with cerebral palsy.