Our aim was to investigate whether rapid changes in visual input or prolonged visual deafferentation modify primary motor cortex (M1) excitability in healthy subjects. rTMS, consisting of 10 stimuli delivered at 5 Hz, at 120% of resting motor threshold was delivered over M1 in 13 healthy volunteers. They were instructed to relax under eyes-opened (EO) and eyes-closed (EC) resting conditions. Two experimental sessions were performed. In the first session, subjects were tested in both EO and EC conditions in order to see whether short visual deprivation affected the M1 excitability, through the possible changes in the motor evoked potential (MEP) amplitude during the rTMS. In the second session, rTMS was delivered both under EO with room lights on and after 30 min of blindfolding in order to evaluate the effects of prolonged visual deprivation on the M1 excitability. A short-term visual deprivation left the MEP facilitation unchanged during 5 Hz-rTMS trains, whilst 30 min of blindfolding significantly decreased the MEP facilitation. The short-term visual deprivation condition did not significantly influence the M1 excitability, whereas prolonged visual deafferentation decreased rTMS-induced MEP facilitation. Prolonged visual deafferentation can significantly modulate motor cortical synaptic plasticity.