Blastocyst implantation is a critical stage in the establishment of pregnancy. Leukemia inhibitory factor (LIF) is essential for mouse blastocyst implantation and also plays a role in human pregnancy. We examined the effect of a potent LIF antagonist (LA) on mouse implantation. In mice, LIF expression peaks on day 3.5 of pregnancy (D3.5) (D0.5 = day of mating plug detection) in the uterine glandular epithelium. LA (7 mg/kg per day) administered from D2.5 to D4.5 via four hourly i.p. injections plus continuous administration via miniosmotic pump resulted in complete implantation failure. To improve its pharmacokinetic properties, we conjugated LA to polyethylene glycol (PEG), achieving a significant increase in serum levels. PEGylated LA (PEGLA) (37.5 mg/kg per day) administered via three i.p. injections between D2.5 and D3.5 also resulted in complete implantation failure. PEGLA immunolocalized to the uterine luminal epithelium at the time of blastocyst implantation. Both LA and PEGLA reduced phosphorylation of the downstream signaling molecule STAT3 in luminal epithelial cells on D3.5. The effects of PEGLA were found to be endometrial, with no embryo-lethal effects observed. These data demonstrate that administration of a PEGylated LIF antagonist is an effective method of targeting LIF signaling in the endometrium and a promising novel approach in the development of nonhormonal contraceptives for women.