We report visible spectroelectrochemical (SEC) characterization of cytochrome c 552 (cyt c 552 ) in viable Geobacter sulfurreducens biofilms on tin-doped indium oxide (ITO) electrodes poised at 0.24V vs. SHE. G. sulfurreducens biofilms were grown in minimal medium with acetate as electron donor (turnover conditions), followed by 24h incubation in electron donor-depleted medium (non-turnover conditions). The electronic absorption spectra of G. sulfurreducens biofilms showed the lowest energy absorption band in the reduced state at 552nm, which indicated excess of cyt c 552 in the biofilm. The spectra under non-turnover conditions displayed gradual reduction of the cyt c 552 , following the step-wise decrease of electrode potential from 0.0V to −0.6V vs. standard calomel electrode (SCE). The spectral changes were fully reversible in both positive and negative direction of the scan potential, with average midpoint potential value of −0.42V vs. SCE. Confocal microscopy analysis revealed that the thickness of biofilms under turnover conditions and non-turnover conditions was approximately 35 and 3.5μm, respectively. This is the first study to observe the reversible redox conversion of cyt c 552 in viable G. sulfurreducens biofilms.