Tetramethylpyrazine (TMP), one of the active ingredients isolated from a Chinese herbal prescription, possesses protective effects against apoptosis in endothelial cells. However, the underlying mechanism of its protective effects in endothelial cells remains to be elucidated. Using human umbilical vein endothelial cells (HUVECs), the present study assessed the protective effects of TMP on CoCl2-induced apoptosis. Following pre-incubation with CoCl2 (150 µM/ml) for 4 h, the HUVECs were treated with TMP at different concentrations (50, 100 and 200 µM/ml) for 8 h. TMP upregulated the expression of prolyl hydroxylase (PHD)2, reduced the protein and mRNA expression levels of vascular endothelial growth factor (VEGF), and reduced the expression of HIF-1α only at the protein level, not at the mRNA level in HUVECs, in a concentration-dependent manner. Furthermore, silencing of the PHD2 gene with small interfering (si)RNAs abolished the reduction in the expression of hypoxia-inducible factor (HIF)-1α and VEGF by TMP. In addition, TMP protected CoCl2-induced HUVEC injury via an apoptosis pathway, as characterized by the increased ratio of cell viability and the reduced percentage of apoptotic and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive HUVECs, activation of caspase-3, -8 and -9, B-cell lymphoma (Bcl)-2/Bcl-2-activated X protein expression, as well as the release of cytochrome c. The protective properties of TMP were partially attributed to the mRNA and protein expression levels of PHD, since silencing of the PHD2 gene with siRNAs abolished these effects. The present study demonstrated that the antiapoptotic effect of TMP in CoCl2-induced HUVECs was, at least in part, via the regulation of the PHD2/HIF-1α signaling pathway.