The extracellular electron transfer (EET) process of Shewanella species is believed to be indispensable for their anaerobic respiration with an electrode. However, the function of outer membrane c-type cytochromes (OM c-Cyts, the primary components of the EET pathway) is still controversial. In this study, we investigated the effect of two OM c-Cyts (MtrC and UndA) of Shewanella putrefaciens CN32 with respect to electricity production and anodic EET efficiency. Deletion of the mtrC gene severely prolonged the microbial fuel cell (MFC) start-up time and decreased electricity production due to depressed flavin-mediated electron transfer, whereas deletion of the undA gene did not have a significant impact. Strikingly, the depression of EET by the deletion of mtrC could be partially relieved by acclimation, which might be due to an increase in the transmembrane transport of electron shuttles and/or the activation of other redox proteins. These results suggested that MtrC may be the primary reductase of flavins to ensure fast indirect EET, which plays a crucial role in MFC electricity generation.