Aims: Upregulation of substance P (SP) and neurokinin-1 receptor (NK1R) activation induces pro-inflammatory bladder hyperactivity through the PKC/ERK/NF-kappa B/ICAM-1/IL-33 signaling pathways to increase the leukocyte infiltration and adhesion leading to reactive oxygen species (ROS) production, autophagy, and apoptosis. L-Theanine is a unique non-protein-forming amino acid present in tea (Camellia sinensis [ L.] O. Kuntze) with its antioxidant, antiinflammatory, and relaxation effects to improve cognition, mood, gastric ulcer injury, and cerebral ischemia/reperfusion injury, and posttraumatic stress disorder. We explored the protective effect of L-theanine on SP-induced bladder hyperactivity. Methods: In urethane-anesthetized female Wistar rats, we explored the transcystometrogram, pelvic nerve activity, proinflammatory PKC/ERK/NF-kB/ICAM-1/IL-33 signaling, apoptosis-related Caspase 3/poly-(ADPribose)- polymerase (PARP), and autophagy-mediated LC3 II expression by Western blot, electrophoretic-mobility shift assay and immunohistochemistry, bladder ROS amount by a ultrasensitive chemiluminescence method, and possible ROS sources from the different leukocytes by specific stains in SP-evoked hyperactive bladder. Results: L-Theanine dose-dependently depressed H2O2 and HOCl activity in vitro. In urethane-anesthetized female Wistar rats, intra-arterial SP through NK1R activation increased voiding frequency (shortened intercontraction intervals) associated with the increase in bladder nerve activity, proinflammatory PKC/ERK/NF-kB/ICAM-1/IL-33 signaling, Caspase 3/PARP-mediated apoptosis, LC3 II-mediated autophagy, ROS amount, neutrophils adhesion, CD68 (monocyte/macrophage) infiltration, and mast cells degranulation in the hyperactive bladder. Intragastrical L-theanine (15 mg/kg) twice daily for 2 weeks efficiently ameliorated all the enhanced parameters in the SP-treated hyperactive bladder. Conclusions: In conclusion, L-theanine through antioxidant and anti-inflammatory actions ameliorates SP-induced bladder hyperactivity via the inhibition of proinflammatory PKC/ERK/NF-kB/ICAM-1/IL-33 signaling, oxidative stress, bladder nerve hyperactivity, apoptosis, and autophagy. (C) 2016 Wiley Periodicals, Inc.