In ruminants, prostaglandin F2 alpha (PGF2_alpha) is synthesized and released in a pulsatile pattern from the endometria luminal epithelial (LE) cells during the process of luteolysis. Prostaglandin transporter (PGT) is a 12-transmembrane solute carrier organic anion transporter protein that facilitates transport of PGF2_alpha. The present study determined the effects of inhibition of PGT protein on pulsatile release of luteolytic PGF2_alpha and the underlined cell-signaling mechanisms. The results indicate that intrauterine inhibition of the PGT protein inhibits the pulsatile release of PGF2_alpha from the endometrium and maintains a functional corpus luteum. Surprisingly, inhibition of PGT-mediated luteolytic pulses is not associated with spatial regulation of estrogen and oxytocin receptors in the LE of the endometrium and is also not accompanied by decreased biosynthesis of PGF2_alpha or increased catabolism of PGF2_alpha by the endometrium. Importantly, PGT inhibitor increases expression of pERK1/2 proteins in the LE of the endometrium. Knock down of ERK1/2 genes in LE cells reverses the inhibitory effects of PGT inhibitor on release of PGF2_alpha. In conclusion, intrauterine inhibition of PGT inhibits the pulsatile release of PGF2_alpha from the endometrium without modulating spatial expressions of estrogen and oxytocin receptor proteins and metabolism of PGF2_alpha at the time of luteolysis. Activation of ERK1/2 pathways and interactions between ERK1/2 and PGT protein appear to be important cell-signaling mechanisms that control PGT-mediated efflux transport function. PGT emerges as an important final component in the luteolytic machinery that controls the release of luteolytic pulses of PGF2_alpha from the endometrium in sheep.