BACKGROUND L-theanine is a non-protein amino acid in green tea, and its hepatoprotection and neuroprotection have been verified. However, whether L-theanine can prevent cardiomyocytes from apoptosis is unclear yet. This study evaluated the protective effects of L-theanine on H2O2-induced heart injury in vitro. MATERIAL AND METHODS The certified H9C2 cells were pretreated with L-theanine (0 mM, 4 mM, 8 mM, and 16 mM) for 24 h, followed by 160 µM H2O2 solution for 4 h. The cell viability and antioxidant indices were assayed. Quantitative evaluation of apoptosis was performed by flow cytometric analysis. Nuclear morphology of the cells was monitored by 4',6-diamidino-2-phenylindole staining. Expression of Caspase-3, poly ADP-ribose polymerase (PARP), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase p38 was assayed by Western blot. RESULTS Compared to the H2O2 treatment, all doses of L-theanine treatments increased the cell viability, glutathione level, and the activities of glutathione peroxidase and superoxide dismutase (P<0.001). The contents of reactive oxygen species, nitric oxide, and oxidized glutathione were decreased by L-theanine treatments (P<0.001). Meanwhile, L-theanine treatments decreased the apoptosis ratio of H2O2-induced H9C2 cells (P<0.001). Pro-Caspase-3 expression was upregulated and cleavaged-PARP expression was inhibited by L-theanine (P<0.001). However, the phosphorylation of JNK and p38 was not affected by L-theanine treatments (P>0.05). CONCLUSIONS These data indicate that L-theanine pretreatment prevents H2O2-induced apoptosis in H9C2 cells, probably via antioxidant capacity improvement. Therefore, it might be a promising potential drug candidate for prophylaxis of ischemia/reperfusion-induced heart diseases.