Background <p>Influenza A viruses (IAVs) initially infect a few host cells before spreading to neighboring cells. However, the molecular mechanisms underlying this dissemination remain unclear. We have previously demonstrated that intracellular Ca²⁺ plays a crucial role in facilitating IAV infection. This study aims to clarify the connections between intracellular Ca²⁺ dynamics and spread of IAV infection.</p> Methods <p>Madin–Darby canine kidney (MDCK) cells stably expressing a Ca²⁺ indicator for optical imaging were established. Cells were cultured in Matrigel to form monolayers, and cell-to-cell Ca²⁺ dynamics within IAV-infected cells were analyzed using fluorescence microscopy.</p> Results <p>IAV infection upregulated the frequency of intercellular calcium wave propagations (iCWPs), facilitating viral spread. ADP released from initially infected cells mediated iCWPs via the P2Y₁ receptor. P2Y₁ antagonist suppressed both the generation of iCWPs and spread of viral infection. Enhanced endocytosis by the surrounding cells that received ADP signaling upregulated viral entry. Expression of IAV matrix protein 2 (M2) in initially infected cells triggered iCWPs through ADP diffusion, thereby increasing infection. Conversely, an ion permeability-deficient mutation of M2 or inhibition of its ion channel activity suppressed iCWPs.</p> Conclusions <p>Intercellular calcium signaling plays a crucial role in the early expansion and establishment of IAV infection, presenting a potential target for IAV prophylaxis.</p>