A variety of pulsed electromagnetic fields (PEMFs) have been experimentally and clinically used in an effort to promote wound healing, although the mechanisms involved remain unknown. The aim of the present study was to investigate the action of a novel protocol of co-treatment with PEMFs and hydroxytyrosol (HTY) on the proliferation and differentiation potential of human umbilical vein endothelial cells (HUVECs). The HUVECs were assigned randomly into three groups: Control, PEMF-treated and PEMF + HT-treated. The intensity of the electromagnetic field used in this protocol was 2.25 mT, the frequency of the bursts was 50 Hz and the application time was 15 min. A Cell Counting kit-8 (CCK-8) assay was used to assess cell proliferation, and cell apoptosis was analyzed by TUNEL apoptosis assay kit and calcein-acetoxymethyl/propidium iodide dual-staining assay. In addition, protein and mRNA expression levels of protein kinase B (Akt), mechanistic target of rapamycin (mTOR), transforming growth factor (TGF)-β1 and p53 were determined by western blotting and reverse transcription-quantitative polymerase chain reaction assays, respectively. The CCK-8 assay demonstrated that HTY contributed to HUVEC proliferation mediated by PEMFs in a time-dependent manner. The Transwell assay and scratch wound results demonstrated that co-treatment of HTY and PEMFs could increase HUVEC migration. Furthermore, the levels of apoptotic cells were reversed by pre-incubation with HTY in the PEMF treatment group, while PEMF treatment alone had no such effect. The proteins and mRNA expression levels of Akt, mTOR, TGF-β1 were elevated in co-treatment of HTY and PEMFs, whereas there was no effect on levels of p53. Therefore, the results indicated that combined exposure of HUVECs to PEMFs and HTY exerted protective effects in HUVECs by promoting cell proliferation and inhibiting apoptosis. In conclusion, to the best of our knowledge, the present study was the first to demonstrate the beneficial roles of HTY and PEMF combined treatment in HUVECs, which may represent an effective treatment for wound healing.