Abstract: Bacteriorhodopsin（bR）, a seven-transmembrane photoreceptor protein with retinal as the chromophore found in purple membrane of Halobacterium salinarum R1M1, which has proton pump function. Tryptophan 182（W182）, one of the most conserved aromatic residues within the microbial rhodopsin family proteins, locates in the intracellular side of the retinal binding pocket and plays a very important role in affecting the photocycle and proton pumping activity of bR through the interaction with the retinal polyene chain. In this study, site-directed mutagenesis of W182 F and W182 A, in combination with in-situ UV-vis absorption spectroscopy, light-induced transient absorption change spectroscopy, pH titration and molecular dynamics simulations, were employed to elaborate the functional role of W182 in the bR photocycle. Our results showed that the mutation of W182 F caused a great disturbance to the binding region of retinal, while the mutation of W182 A had a great effect on the intracellular half channel of the protein. The two mutations had different molecular mechanisms on the effect of proton transport on the extracellular half channel, resulting in a significant difference in the decay of M intermediate state.