During pregnancy, disorders in uterine spiral artery remodeling (USAR) cause preeclampsia and other diseases. The aim of this study was to investigate the effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on the biological behavior of human villous trophoblasts (HVTs) and human uterine vascular smooth muscle cells (HUVSMCs), and explore the role of 20-HETE in USAR. 20-HETE and its inhibitor HET0016 were used to study migration, invasion and apoptosis in the HVT and HVT-HUVSMC models. 20-HETE inhibited the migration and invasion of HVTs, and inhibited apoptosis in HUVSMCs and HUVSMCs co-cultured with HVTs. 20-HETE had thus obvious effects on the biological behavior of HVTs and HUVSMCs. These effects may cause USAR disorders and vascular dysfunction, leading to preeclampsia.