The goal of the current study was to examine the role of the ubiquitin-proteasome system (UPS), a pathway of intracellular degradation, in the regulation of fetal fibronectin (FFN) expression in human placenta. Primary cultures of cytotrophoblasts (CTs) and placental mesenchymal cells (PMCs) were isolated from human term placentas and were maintained in serum-free medium (SFM) in the presence of inhibitors of proteasome-mediated degradation (e.g., MG132) as well as inhibitors of other proteases. Levels of secreted FFN and interleukin (IL)-8 in culture media were quantitated by enzyme-linked immunosorbent assay (ELISA), and cell viability was assessed by trypan blue exclusion. Intracellular levels of FFN and ubiquinated proteins were measured by Western blotting, and levels of fibronectin mRNA were determined following Northern blotting. We found that proteasome inhibitors (MG132, MG262, and PSI) potently suppressed levels of secreted FFN in cultures of CTs, but they not did affect levels of IL-8. Lysosomal, calpain, and serine protease inhibitors as well as the anti-inflammatory compound sulfasalazine did not markedly affect levels of secreted FFN in CT cultures. Proteasome inhibitors did not compromise cell viability during the initial 16–18 hours of treatment and did not affect intracellular levels of FFN protein or fibronectin mRNA. The efficacy of suppression of FFN in CT culture media by proteasome inhibitors reflected their effects on intracellular accumulation of ubiquinated proteins. By contrast, the presence of proteasome inhibitors did not alter levels of secreted FFN in cultures of PMCs. We conclude that inhibitors of proteasome-mediated degradation potently and specifically suppressed extracellular expression of FFN in CTs through a cell type-specific pathway that did not involve alterations in FFN synthesis. This suggests that accumulation of ubiquinated proteins in the presence of proteasome inhibitors blocks FFN secretion or promotes the extracellular degradation of FFN. This experimental paradigm will be useful for dissecting the role of the UPS in regulating CT function.