The effect of testosterone, a sex steroid, on endothelial cells is controversial as it is uncertain if it has a protective effect on them. Whether physiological testosterone can inhibit the deleterious effects of advanced glycation end products (AGEs) on endothelial cells remains to be elucidated. The present study focused on elucidating the effect of testosterone on the injury of endothelial cells induced by AGEs. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with AGEs in the presence or absence of various concentrations of testosterone. The cell viability in each group was measured using an MTS assay. Early‑stage apoptosis was detected using flow cytometry with Annexin V‑fluorescein isothiocyanate/propidium iodide double staining, and the expression levels of apoptosis‑associated proteins, B cell lymphoma‑2 (Bcl‑2), Bcl‑2‑associated X protein (Bax) and caspase‑3, were determined using western blot analysis. Oxidative stress and pro‑inflammatory parameters in the medium were evaluated using an enzyme‑linked immunosorbent assay. The MTS results showed that AGEs significantly decreased the proliferation of HUVECs, whereas a physiological concentration of testosterone alleviated this damage. Physiological concentrations of testosterone protected the HUVECs from AGE‑induced apoptosis, mediated by caspase‑3 and Bax/Bcl‑2. In addition, treatment of the HUVECs with AGEs caused a significant decrease in anti‑oxidative parameters, but increased the concentrations of malondialdehyde and tumor necrosis factor‑α. The activation of Janus kinase 2 and signal transducer and activator of transcription 3 was significantly increased by incubation with AGEs. However, pre‑incubation with a physiological concentration of testosterone attenuated these changes. Therefore, the data obtained in the present study established the potential role of physiological testosterone in ameliorating AGE‑induced damage in HUVECs.