Significance The influenza A M2 channel (AM2) transports protons into the influenza virus upon acid activation. It is an important pharmacological target as well as a prototypical case to study proton conduction through biological channels. The current work provides the most complete computational characterization to date of the physical basis for the acid activation mechanism of the AM2 proton channel. Our results show that lowering the pH value gradually opens the Trp41 gate and decreases the deprotonation barrier of the His37 tetrad, leading to channel activation. Our result also demonstrates that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain.