Electrogenerate bacteria were a kind of current-producing bacteria, which possess the ability to generate and transport electrons. The mechanism of its extracellular electron transfer is a key scientific problem in bioelectrochemical system. Till to now, due to obscure understanding toward this problem, it directly affects the energy conversion efficiency of biological electrochemical system and restricts the application in energy conversion and environmental remediation and management. At present, it was reported that there were three kinds of possible electron transport pathways : (1) electrons could be transferred from series proteins in the cell membrane to the final electron acceptor; (2) electrons could be indirectly transferred by electron mediators or shuttles to electron acceptor, for example, riboflavin, a metabolite from S. oneidensis MR-1; (3) electron could be transferred by electrically conductive pili to electron acceptor. In this work, Shewanella oneidensis (S. oneidensis) MR-1, a Gram-negative bacterium, was used as a target bacterium, cyclic voltammetry (CV) and electrochemical in situ FTIR spectroscopy (in situ FTIRS) in water and deuterated water solution were used to study the effect of OmcA-MtrC protein in the electron-transfer processes by comparing Shewanella oneidensis (S. oneidensis) MR-1 (wild type) with △omcA-△mtrC mutant. The CV and in situ FTIRS results illustrate that △omcA-△mtrC mutant produced by gene knockout of OmcA-MtrC protein from wild bacteria still possesses electron transfer capability, but weakly than wild type. The results suggested that another electron transfer pathway existed indeed besides by OmcA-MtrC protein. The current study illustrated that electrochemical in situ FTIRS is an effective approach in investigating electron transfer process of bacteria, and shed lights on the study of the bacterial bioactivity.