Influenza A virus can evade host innate immune response that is involved in several viral proteins with complicated mechanisms. To date, how influenza A M2 protein modulates the host innate immunity remains unclear. Herein, we showed that M2 protein colocalized and interacted with MAVS (mitochondrial antiviral signaling protein) on mitochondria, and positively regulated MAVS-mediated innate immunity. Further studies revealed that M2 induced reactive oxygen species (ROS) production that was required for activation of macroautophagy/autophagy and enhancement of MAVS signaling pathway. Importantly, the proton channel activity of M2 protein was demonstrated to be essential for ROS production and antagonizing the autophagy pathway to control MAVS aggregation, thereby enhancing MAVS signal activity. In conclusion, our studies provided novel insights into mechanisms of M2 protein in modulating host antiviral immunity and uncovered a new mechanism into biology and pathogenicity of influenza A virus.