Abstract: Bacteriorhodopsin（bR） is a light-driven photoreceptor. Each active unit is made up of three monomers and each monomer contains one opsin molecule and one retinal（Ret） chromophore covalently bonded to each other,functioning as a proton pump to transport protons from the cytoplasmic side to the extracellular side directionally to convert light energy into chemical energy through formed proton concentration gradient. Photo-isomerization of the Ret chromophore from the all-trans to the 13-cis configuration triggers cyclic conformation changes that include a series of stable photo-intermediates to transfer a proton vectorial across the cell membrane. Tryptophan 86（W86）, one of the key aromatic amino acid residues in the Ret binding pocket, is highly conservative in all microbial rhodopsin family proteins. To reveal the mechanism of the coupling of W86 with the bR photocycle, in this study, in situ solid-state NMR correlation experiments, combined with light-induced transient absorption change measurements, p H titration and site-specific mutation, were employed to elucidate the functional roles of W86 during the bR photocyc. In order to investigate the effect of Tryptophan 86（W86）, a conserved amino acid in BR retinal bond region, on its photocyclic intermediate state and proton pump function, we mutated W86 into F86 and A86 with different side chain sizes by site mutation technique. In situ UV-Vis absorption spectroscopy, flash photolysis spectroscopy, pH titration, solid state nuclear magnetic resonance and other techniques were used to explore the molecular mechanism of the difference between W86 F and W86 A mutations on BR photocirculation and proton pump functions. The results showed that both W86 F and W86 A mutations shift the dark-adapted isomerization thermal equilibrium of Ret towards a cis dominant direction with the all-trans isomer completely disappearing in W86 F. In addition, both W86 F and W86 A mutations lead to weakened and prolonged attenuation of the intermediate state of the protein photocycle, as well as weakened proton pump function, but the influencing mechanisms are different, which may be related to the difference in the distribution of electron clouds on the retinal-polyene chain and the degree of disturbance caused by the surrounding residues.