Gamete and embryo transport is an important function of the oviduct. This type of transport involves both smooth muscle contraction and epithelial cell secretions, and the former is mediated by prostaglandins (PGs) and their receptors. Our objective was to study the regulation of prostaglandin synthetase (prostaglandin-endoperoxide synthase-1 (PTGS1), prostaglandin-endoperoxide synthase-2 (PTGS2), mPGES-1, mPGES-2, cPGES, and PGFS) by estradiol (E2) in bovine oviduct smooth muscle. Prostaglandin synthetase mRNA and protein expression were investigated using real-time RT-PCR and Western blot analyses, respectively. Prostaglandin synthetase mRNA and protein expression were increased in oviductal smooth muscle tissue after treatment with different concentrations of estradiol for various time periods. The results indicated that there was no increase in expression observed after treatment with fulvestrant, a selective antagonist of the E2 receptor, indicating that E2 interacts with specific E2 nuclear receptors to upregulate PTGS1, PTGS2, mPGES-1, and PGFS expression. In conclusion, E2 increases PTGS1, mPGES-1, and PGFS mRNA and protein expression in bovine oviductal smooth muscle when added for different periods of time and at different concentrations. Additionally, E2 is transported intracellularly and interacts with specific E2 nuclear receptors to increase PTGS1, PTGS2, mPGES-1 and PGFS expression.