The related sulfur oxidation membrane protein genes of extremely thermophilic archaea Acidianus manzaensis were screened and identified based on comparative proteomics methods. Firstly, membrane proteins of A. manzaensis grown on two energy sources, elemental sulfur (S0) and ferrous ions (Fe2+) were selectively extracted and primitively validated by temperature-induced two-phase separation technique of Triton X-114 and real time polymerase chain reaction (RT-PCR). The extracted membrane proteins were further analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). Finally the membrane proteins expressed spots under S0 substrate were selected to identify by tandem time of flight mass spectrometry (MALDI-TOF/TOF) and then were verified by RT-qPCR. The results of the present study indicated that the membrane proteins extracted from the two energy sources were significantly different. One hypothetical protein was found rich in cysteine residues and had one —CXXC— functional motif in eight related sulfur oxidation membrane protein genes, which might play an important role in the sulfur transportation by forming the Pr—SSnH（n≥2）complex using the thiol groups (—SH) to bind the S0. Sulphide-quinone oxidoreductas, electrons transportation proteins, transcriptional regulation proteins and grown relevant proteins were also identified, these membrane proteins might play important roles with thermophilic archaea Acidianus manzaensis during S0 oxidation process.