The AAA ATPase spastin is a microtubule-severing enzyme that plays important roles in various cellular events including axon regeneration. Herein, we found that the basal ATPase activity of spastin is negatively regulated by spastin concentration. By determining a spastin crystal structure, we demonstrated the necessity of inter-subunit interactions between spastin AAA domains. Neutralization of the positive charges in the microtubule-binding domain (MTBD) of spastin dramatically decreased the ATPase activity at low concentration, although the ATP-hydrolyzing potential was not affected. These results demonstrate that, in addition to the AAA domain, the MTBD region of spastin is also involved in regulating ATPase activity, making interactions between spastin protomers more complicated than expected.