Impaired airway innate immunity (e.g., suppressed Toll-like receptor 2 [TLR2] signaling) has been reported in allergic lungs with bacterial infection. Recently, an allergic mouse lung milieu including the T-helper type 2 (Th2) cytokine interleukin-13 (IL-13) has been shown to up-regulate lung microRNA-21 (miR-21) expression. Whether miR-21 modulates in vivo TLR2 signaling is unknown. The goal of this study was to determine if in vivo, miR-21 regulates a TLR2 agonist-induced lung inflammatory response. Balb/c mice were intranasally pretreated with a locked nucleic acid (LNA) in vivo inhibitor probe for mouse miR-21 or a control probe, followed by intranasal instillation of a TLR2 agonist Pam3CSK4, or saline (control). Four and/or 24 hours later, mice treated with the miR-21 inhibitor probe, as compared to the control probe, significantly increased lung leukocytes, including neutrophils and the keratinocyte-derived chemokine (KC). IL-13 treatment for 72 hours increased (P < .05) miR-21 in cultured primary normal human airway epithelial cells. These results, for the first time, suggest an in vivo role of miR-21 in suppressing TLR2 signaling, and further support that IL-13 can up-regulate miR-21 in human airway epithelial cells. Functional studies on miR-21 likely provide novel approaches to modulate TLR2 signaling in Th2 cytokine-exposed airways.