Human neuroglobin (Ngb) is a heme protein expressed in the brain tissue and retina with an important neuroprotective role. However, its molecular mechanism is still under exploration. To gain further insight into this issue, we developed an electrochemical strategy using a facilely prepared nanoporous gold (NPG) electrode. Immobilization of Ngb onto the NPG surface enabled rapid direct electron transfer between Ngb and the electrode, whereas less electrochemical response was generated using a planar gold electrode. Voltammograms recorded using the NPG electrodes gave an E-0' value of -0.170 V (vs. standard hydrogen electrode (SHE)), which is in good agreement with the previously reported values. Furthermore, a heterogeneous electron transfer rate constant of 46 s(-1) was obtained, which was larger than the rate constants reported previously. The addition of ferric cytochrome c (Cyt c), a potential redox partner protein of Ngb, increased the cathodic current in the voltammogram, indicating the electron transfer from the reduced (ferrous) Ngb to the ferric Cyt c. In addition, mutational studies demonstrated for the first time that the disulfide bond formation between Cys46 and Cys55 is important for this electron transfer. These results may indicate that ferrous Ngb, in addition to the ferric form, also contributes to the anti-apoptotic properties of Ngb, depending on the disulfide bond formation. Thus, the NPG-based electrochemical system developed in our study is an effective tool for uncovering the molecular basis of Ngb coupled with electron transfer.