Two-dimensional infrared (2D IR) spectroscopy, introduced two decades ago, has been developed rapidly and been widely used to investigate the structure and fast dynamics of small molecules to large biomolecules such as proteins. As an example, a 2D IR study of the pH-dependent structure and hydrogen-bond dynamics of the M2 proton channel embedded in the supported lipid bilayers is presented here. The M2 is one of the smallest proton-selective channels found in nature and is the ideal system for studying the use of water when selectively transporting protons across the membrane. To better understand the mechanism of membrane protein function, it is essential to investigate the interaction with the environment. Among many interactions, the hydrogen-bonding interaction between membrane proteins and water molecules is crucial to understanding the role of water molecules in the membrane protein pores for the transport of substrates across membrane bilayers. The M2 proton channel exhibits a higher content of water molecules in the channel cavity at lower pH (5.5) than at higher pH (7.5), which was confirmed by the water hydrogen-bond exchange between the protein and water. Changes in the backbone structure accompanied by the pH change were also observed by polarization-dependent 2D IR experiments.