Motor axon regeneration following peripheral nerve injury is critical for motor recovery but therapeutic interventions enhancing this are not available. We conducted a phenotypic screen on human motor neurons and identified blebbistatin, a non-muscle myosin II inhibitor, as the most effective neurite outgrowth promotor. Despite its efficacy in vitro, its poor bioavailability limits in vivo application. We, therefore, utilized a blebbistatin analog, NMIIi2, to explore its therapeutic potential for promoting axon regeneration. Local NMIIi2 application directly to injured axons enhanced regeneration in human and mouse motor neurons in vitro. Furthermore, following a sciatic nerve crush injury in mice, local NMIIi2 administration to the axonal injury site facilitated motor axon outgrowth, muscle reinnervation, and functional recovery. NMIIi2 also promoted axon regeneration in sensory, cortical, and retinal ganglion neurons. These findings highlight the therapeutic potential of topical NMII inhibition for treating axon damage.