Human Cytomegalovirus (HCMV) is a widespread double-stranded DNA virus, which can cause serious complications in fetuses and neonates after infection by pregnant women. HCMV can synthesize a variety of glycoproteins, among which gpUL132,a 270-amino-acid type I envelope glycoprotein has attracted more and more attention because it was found to be involved in the formation of viral assembly compartment in recent years. Human placental trophoblast cells, as the first layer of placental barrier cells, can play an antiviral role after pathogen invasion. However, whether gpUL132 is involved in HCMV infection of human placental trophoblast cells and its specific mechanism is still unclear. In this study, we found gpUL132 protein of HCMV could activate the nuclear factor-kappa B (NF-κB) pathway induced by polyinosinic-polycytidylic acid [poly(I:C)], a synthetic analog of double-stranded RNA. Poly(I:C) treatment induced a higher level of P65 phosphorylation in HPT-8 cells expressing gpUL132 as compared to control cells. Additionally, HCMV gpUL132 activated NF-κB P65 via the toll-interleukin 1 receptor homology-domain-containing adapter-inducing interferon-β (TRIF)-dependent TLR3 pathway. Finally, gpUL132 protein could increase the TRIF level by extending its half-life.