This study aims to explore the effects of arachidonic acid lipoxygenase metabolism in vascular calcification. We used 5/6 nephrectomy and high-phosphorus feeding to establish a model of vascular calcification in mice. Six weeks after nephrectomy surgery, vascular calcium content was measured, and Alizarin Red S and Von Kossa staining were applied to detect calcium deposition in aortic arch. Control aortas and calcified aortas were collected for mass spectrometry detection of arachidonic acid metabolites, and active molecules in lipoxygenase pathway were analyzed. Real-time quantitative PCR was used to detect changes in the expression of lipoxygenase in calcified aortas. Lipoxygenase inhibitor was used to clarify the effect of lipoxygenase metabolic pathways on vascular calcification. The results showed that 6 weeks after nephrectomy surgery, the aortic calcium content of the surgery group was significantly higher than that of the sham group (P < 0.05). Alizarin Red S staining and Von Kossa staining showed obvious calcium deposition in aortic arch from surgery group, indicating formation of vascular calcification. Nine arachidonic acid lipoxygenase metabolites were quantitated using liquid chromatography/mass spectrometry (LC-MS) analysis. The content of multiple metabolites (12-HETE, 11-HETE, 15-HETE, etc.) was significantly increased in calcified aortas, and the most abundant and up-regulated metabolite was 12-HETE. Furthermore, we examined the mRNA levels of metabolic enzymes that produce 12-HETE in calcified blood vessels and found the expression of arachidonate lipoxygenase-15 (Alox15) was increased. Blocking Alox15/12-HETE by Alox15 specific inhibitor PD146176 significantly decreased the plasma 12-HETE content, promoted calcium deposition in aortic arch and increased vascular calcium content. These results suggest that the metabolism of arachidonic acid lipoxygenase is activated in calcified aorta, and the Alox15/12-HETE signaling pathway may play a protective role in vascular calcification.