Understanding the mechanistic basis of the response of neurons to injury is directly relevant to the development of effective therapeutic approaches aimed at the amelioration of nervous system damage. Axons retract in response to severing. We investigated the mechanism of axon retraction in response to severing in vitro, testing the hypothesis that actomyosin contractility drives severing-induced axon retraction. Axon retraction commenced within 5 min following severing and correlated with actin filament accumulation at the site of severing. Depolymerization of actin filaments prevented retraction, demonstrating that actin filaments are required for severing-induced axon retraction. Direct inhibition of myosin II, using blebbistatin, minimized axon retraction in response to severing. Blocking RhoA-kinase (ROCK), a modulator of myosin II activity, inhibited axon retraction. Similarly, inhibiting myosin light chain kinase (MLCK) with a cell-permeable pseudo-substrate peptide also inhibited axon retraction. These data demonstrate that myosin II activity is required for severing-induced axon retraction in vitro, and suggest myosin II as a target for therapeutic interventions aimed at minimizing retraction following severing in vivo.