Recent decades have witnessed a rapid worldwide increase in chronic inflammatory disorders such as asthma. CD4+ T helper type 2 (Th2) cells play critical roles in the pathogenesis of allergic airway inflammation and CD69 expression on activated CD4 T cells is required to induce allergic inflammation in tissues. However, how CD69 mechanistically controls allergic inflammation remains poorly defined. In lymphoid tissues, CD69 regulates cellular retention via inhibition of S1P1 expression and requires no specific ligands to function. In contrast, we show herein that myosin light chain (Myl) 9 and 12 are new functional ligands for CD69. The blockade of the CD69-Myl9/12 interaction ameliorates allergic airway inflammation in OVA-induced and house dust mite (HDM)-induced mouse models of asthma. Within the inflamed mouse airways, we found that the expression of Myl9/12 was increased and platelet-derived Myl9/12 localized to the luminal surface of blood vessels and formed intravascular net-like structures. The analysis of nasal polyps of eosinophilic chronic rhinosinusitis (ECRS) patients revealed that Myl9/12 expression was increased in the inflammatory lesion, and was distributed within net-like structures in the intravascular space. In addition, we detected Myl9/12 in the perivascular spaces where many CD69+ cells were positioned within Myl9/12 structures. Thus, the Myl9/12-CD69 interaction is a key event in the recruitment of activated CD69+ T cells in inflamed tissues and could be a therapeutic target for intractable airway inflammatory diseases.