Klotho is a novel anti-aging hormone involved in human coronary artery disease. The present study aimed to detect the effects and mechanism of Klotho on cardiomyocytes in a hypoxia/reoxygenation (H/R) model in vitro. Neonatal Sprague-Dawley rat cardiomyocytes were randomly distributed into experimental groups as follows: Control group; H/R group, 4‑h hypoxia followed by 3‑h reoxygenation; and H/R+Klotho group, incubated with 0.1, 0.2 or 0.4 µg/ml Klotho protein for 16 h and then subjected to 4‑h hypoxia/3‑h reoxygenation. In order to evaluate cardiomyocyte damage, cell viability and lactate dehydrogenase (LDH) levels were measured. Cell apoptosis was measured by flow cytometry. The 2',7'-dichlorofluorescein diacetate reagent was used to estimate the intracellular generation of reactive oxygen species (ROS). Immunofluorescence staining was used to test whether Klotho induced decreased nuclear translocation of forkhead box protein O1 (FOXO1). Western blot analysis was performed to detect protein levels of FOXO1, phospho-FOXO1, Akt, phospho-Akt and superoxide dismutase 2 (SOD2). Cell viability was significantly decreased, levels of LDH in the cardiomyocyte culture medium were significantly increased and the apoptotic rate was enhanced in the H/R group when compared with those of the control group. Compared with the H/R group, cell viability of the H/R+Klotho groups was significantly higher (P<0.05). Treatment with Klotho protein resulted in a significant resistance of cardiomyocytes to apoptosis and the release of LDH was decreased. Intracellular ROS levels in the H/R group were significantly elevated above those of the control group (P<0.05). Following treatment with Klotho, intracellular ROS levels were significantly decreased compared with those of the H/R group (P<0.05). Western blot analysis confirmed that Klotho protein treatment increased FOXO1 levels in the nucleus and decreased FOXO1 levels in the cytoplasm. Furthermore, exogenous Klotho protein promoted translocation of FOXO1 from cytoplasm to nucleus. In addition, the administration of Klotho protein suppressed phosphorylation of FOXO1 and Akt, and markedly increased the protein expression levels of SOD2. In conclusion, treatment with Klotho protein had beneficial effects on cardiomyocytes undergoing H/R injury. The mechanism of this effect may be associated with suppressed apoptosis of cardiomyocytes, inhibition of phosphorylation of FOXO1 and Akt as well as suppression of cytoplasm transfer of FOXO1.