Hypoxia/reoxygenation (H/R) induced cardiomyocytes apoptosis is a major factor leading to cardiovascular diseases. In this study, we investigated the protective effect of small molecule antidepressant amitriptyline (AMP) in regulating H/R-induced apoptosis in neonatal mouse cardiomyocyte in culture. Cardiomyocytes of C57BL/6J mice were treated with H/R condition in vitro. Various concentration of AMP was added into culture 2 h prior to H/R conditioning. Cardiomyocyte apoptosis was evaluated by TUNEL assay. AMP induced downstream signaling pathway proteins, including tropomyosin receptor kinase A receptor (TrkA), phosphor-TrkA (p-TrkA), protein kinase B (Akt) and phosphor-Akt (p-Akt) were probed by western blot. TrkA phosphorylation was then blocked by K252a to investigate whether TrkA was functionally involved in the protection of AMP in H/R-injured cardiomyocyte. We found that H/R condition induced significant cardiomyocyte death and apoptosis, whereas AMP pretreatment considerably rescued cardiomyocyte death and apoptosis. Western blot analysis showed AMP activated TrkA signaling pathway through the phosphorylation of TrkA/Akt proteins. We also found that application of K252a inhibited the phosphorylation of TrkA/Akt signaling pathway, and subsequently abolished the protective effect of AMP in H/R-induced apoptosis in cardiomyocyte. Thus, our study revealed that AMP, through the activation of TrkA/Akt signaling pathway, plays a protective role in regulating H/R-induced apoptosis in cardiomyocyte.