Cytochrome c is a type of heme proteins that are widely distributed in living organisms. It consists of heme and apocytochrome c, and has potential applications in bioelectronics, biomedicine and pollutant degradation. However, heterologous overexpression of cytochrome c is still challenging. To date, expression of the cytochrome c from uncultured anaerobic methanotrophic archaea has not been reported, and nothing is known about the function of this cytochrome c. A his tagged cytochrome c was successfully expressed in E. coli by introducing a thrombin at the N-terminus of CytC4 and co-expressing CcmABCDEFGH, which is responsible for the maturation of cytochrome c. Shewanella oneidensis, which naturally has enzymes for cytochrome c maturation, was then used as a host to further increase the expression of CytC4. Indeed, a significantly higher expression of CytC4 was achieved in S. oneidensis when compared with in E. coli. The successful heterologous overexpression of CytC4 will facilitate the exploitation of its physiological functions and biotechnological applications.