Bacteriorhodopsin (BR), a light-driven proton pump, has been studied from structure to function for almost four decades by numerous biochemical and biophysical methods. However, the pathways of proton uptake from the cytoplasmic side and release to the extracellular surface are still not very clear. Archaerhodopsin 4 (AR4), a new member of archaeal rhodopsin in Halobacterium species xz515, shares a high sequence similarity and proton pumping function as BR, but exhibits an opposite temporal order for proton release and uptake at neutral pH. Therefore, comparison of the structure and function of the two proteins may provide a deep understanding of the pathway of proton uptake and release mechanism in BR photocycle. In this work, we have successfully established a reliable method to express functional recombinant AR4 in Halobacterium salinarum with a high yield. A series of mutants have been made to examine how surface charge at the extracellular side and the residues in the transmembrane region may mediate the proton transfer order in the two proteins. Experimental results revealed by the light-induced absorption changes indicate that surface charges and some residues located at the transmembrane region may affect both proton transfer kinetics and M intermediate.