HTRA3 is a newly identified serine peptidase of the mammalian HTRA (high-temperature requirement factor A) family, that is upregulated dramatically during mouse placental development. The current study determined whether HTRA3 was involved in human placentation. During the menstrual cycle, HTRA3 was expressed primarily in the endometrial glands, being significantly upregulated toward the mid- to late secretory phases; prominent expression in the stroma detected only in the decidual cells in the late secretory phase. Thus, overall endometrial HTRA3 expression was highest in the late secretory phase, when the endometrium is prepared for maternal-trophoblast interaction. During the first trimester of pregnancy, both glandular and decidual HTRA3 expression increased further with the decidual upregulation being highly significant. The strong link between HTRA3 expression and endometrial stromal cell decidualization was further established in an in vitro model using primary endometrial stromal cells. HTRA3 was also expressed by certain trophoblast subtypes in the first-trimester placenta: strongly in the villous syncytiotrophoblast, trophoblast shell, and endovascular trophoblast and weakly in the distal portion of the trophoblast cell columns but not in villous cytotrophoblast, the proximal region of the cell columns, or interstitial trophoblast. Upregulation of HTRA3 expression in association with placental development was revealed by a significant elevation of this protein in the maternal serum during the first trimester. We thus propose that HTRA3 is a previously unrecognized factor closely associated with and potentially important for human placentation. This study established crucial groundwork for future investigations toward establishing the physiological roles of HTRA3 in human placentation.