L-theanine, a water-soluble amino acid isolated from green tea (Camellia sinensis), has anti-inflammatory activity, antioxidative properties, and hepatoprotective effects. However, the anti-allergic effect of L-theanine and its underlying molecular mechanisms have not been elucidated. In this study, we investigated the protective effects of L-theanine on asthmatic responses, particularly airway inflammation and oxidative stress modulation in an ovalbumin (OVA)-induced murine model of asthma. Treatment with L-theanine dramatically attenuated the extensive trafficking of inflammatory cells into bronchoalveolar lavage fluid (BALF). Histological studies revealed that L-theanine significantly inhibited OVA-induced mucus production and inflammatory cell infiltration in the respiratory tract and blood vessels. L-theanine administration also significantly decreased the production of IgE, monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-4, IL-5, IL-13, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (INF-γ) in BALF. The lung weight decreased with L-theanine administration. L-theanine also markedly attenuated the OVA-induced generation of reactive oxygen species and the activation of nuclear factor kappa B (NF-κB) and matrix metalloprotease-9 in BALF. Moreover, L-theanine reduced the TNF-α-induced NF-κB activation in A549 cells. Together, these results suggest that L-theanine alleviates airway inflammation in asthma, which likely occurs via the oxidative stress-responsive NF-κB pathway, highlighting its potential as a useful therapeutic agent for asthma management.