The Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) represents the first discovered member of enzymes regulated by a voltage-sensor domain (VSD) related to the VSD found in voltage-gated ion channels. Although the VSD operation in Ci-VSP exhibits original voltage dependence and kinetics compared to ion channels, it has been poorly investigated. Here, we show that the kinetics and voltage dependence of VSD movement in Ci-VSP can be tuned over 2 orders of magnitude and shifted over 120 mV, respectively, by the size of a conserved isoleucine (I126) in the S1 segment, thus indicating the importance of this residue in Ci-VSP activation. Mutations of the conserved Phe in the S2 segment (F161) do not significantly perturb the voltage dependence of the VSD movement, suggesting a unique voltage sensing mechanism in Ci-VSP.