The objectives of this study were to determine the effects of flunixin meglumine (FM), a nonsteroidal anti-inflammatory drug on progtaglandin F2alpha (PGF) secretion, by characterizing plasma prostaglandin metabolites (PGFM), and luteal function by characterizing plasma progesterone (P4) concentrations in lactating Holstein dairy cows during the luteal phase of the estrous cycle. On day -35, estrous cycles were synchronized using a Presynch-Ovsynch protocol. Transrectal ultrasonography was performed on days -9, 0, 3, 7, and 15 to confirm ovulation and formation of a corpus luteum (CL). On day 15, cows were stratified by parity and randomly assigned to either treatment or control groups. The treatment group (n=9) received a total of 2.0 mg/kg body weight of FM (iv), whereas the control group (n=8) received saline. Jugular blood samples were collected at 30 and 0 minutes before treatment for PGFM concentrations. Following treatment, blood samples were collected at 30 minutes and every hour for additional 7 hours. Blood samples were also collected daily from days 15 to 22 to characterize P4 concentrations. Plasma PGFM concentrations did not differ between groups before treatment. Following treatment, plasma PGFM remained unchanged in the control group, whereas the mean PGFM concentrations decreased in FM-treated cows (P<0.05). Mean PGFM concentration decreased within 60 minutes following treatment and remained low through the experimental period. Mean P4 concentrations before treatment (day 15) did not differ between the control and treatment groups. Mean P4 concentrations decreased between days 16 to 22. However, the rate of P4 decline over time tended to be smaller for FM-treated cows compared with controls (P=0.09). These results suggest that FM treatment decreases plasma PGFM and therefore, may inhibit PGF secretion in lactating dairy cows during the luteal phase of the estrous cycle. Moreover, FM may help to extend luteal function by reducing the rate of P4 decline.