Embryonic mortality in cattle may occur because of inadequate inhibition of uterine secretion of prostaglandin (PG) F_2α mediated by bovine interferon-τ (bIFN-τ). The objectives of the present study were to determine whether polyunsaturated fatty acids inhibit secretion of PGF_2α from bovine endometrial cells induced by stimulating protein kinase C with phorbol 12,13 dibutyrate (PDBu) and to investigate possible mechanisms of action. Confluent cells were exposed for 24 h to 100 μM of linoleic, arachidonic (AA; C20:4, n-6), linolenic (LNA; C18:3, n-3), eicosapentaenoic (EPA; C20:5, n-3), or docosahexaenoic (DHA; C22:6, n-3) acid. After incubation, cells were washed and stimulated with PDBu. The EPA, DHA, and LNA attenuated secretion of PGF_2α in response to PDBu. The EPA and DHA were more potent inhibitors than LNA. The EPA inhibited secretion of PGF_2α at 6.25 μM. Secretion of PGF_2α in response to PDBu decreased with increasing incubation time with EPA. Both bIFN-τ and EPA inhibited secretion of PGF_2α, and their inhibitory effects were additive. The bIFN-τ, but not EPA, reduced the abundance of PG endoperoxide synthase-2 (PGHS-2) mRNA. Incubation with 100 μM EPA, DHA, or AA for 24 h followed by treatment with PDBu did not affect concentrations of PGHS-2 and phospholipase A₂ proteins. The EPA and DHA inhibit secretion of PGF_2α through a mechanism different from that of bIFN-τ. The effect of EPA on PGF_2α secretion may be caused by competition with AA for PGHS-2 activity or reduction of PGHS-2 activity. The use of EPA and DHA to inhibit uterine secretion of PGF_2α and to improve embryonic survival in cattle warrants further investigation.