Hypertensive disorder of pregnancy (HDP) affects about 10% of pregnant women, which may be caused by dysfunction of placental trophoblasts following impaired uterine spiral arterioles. Our previous analysis revealed that endoplasmic reticulum (ER) stress signaling may be altered in HDP, compared to normal pregnancy. In addition, the expression of high-temperature requirement A1 (HTRA1), a serine protease was decreased in the HDP placenta. However, a role of ER stress and HTRAs in HDP pathophysiology remains unknown. The relationship between of ER stress and HTRAs was explored in vitro human trophoblast cells. Treatment with ER stress inducers thapsigargin or tunicamycin increased the expression of HTRA1 and its subtype HTRA3, but did not alter HTRA2 and HTRA4 in trophoblasts. In vitro invasion assay revealed that either thapsigargin or tunicamycin treatment and the knockdown of HTRA1 or HTRA3 inhibited trophoblast invasion. In addition, ER stress inducers or knockdown of HTRA1 altered the ratio of soluble fms-like tyrosine kinase-1 (sFLT1) to placental growth factor (PGF), a severity index of HDP. The expression of HTRA1 was lower in HDP placenta compared to the normal placenta tissues. These results suggest that ER stress may regulate trophoblast invasion partly via HTRA1 and HTRA3 and is involved in the pathogenic mechanism of HDP. It might be possible to develop a therapeutic means that targets HTRA1 to improve pregnancy complications such as HDP.