Here, the authors report the discovery of a redox switch for AcrIIC1, which regulates AcrIIC1’s monomer-dimer interconversion and inhibitory activity on Cas9. By reporting the structures of different redox states of NmeAcrIIC1, they identify a pair of conserved cysteines which mediate the redox switch. The replacement of the redox-sensitive cysteines allows the generation of a more stable and robust variant under various cellular environments.