Labour is an inflammatory process, with normal on-time delivery requiring leukocyte recruitment and increased cytokine expression in the reproductive and gestational tissues. However, the initiation of these inflammatory events is poorly understood. We hypothesised that on-time labour may involve endogenous ligands of toll-like receptor 4 (TLR4) released from the reproductive and gestational tissues in late pregnancy, and that TLR4 may play an important role in the initiation of the inflammatory response leading to parturition. Mice with a null mutation in the Tlr4 gene (Tlr4-/-) and wild type controls (WT) were mated and gestation length and perinatal parameters were recorded. In a separate experiment uterus, placenta and fetal tissues were recovered from pregnant WT and Tlr4-/- mice on gestational day 16.5, 17.5, 18.5 and 19.5 and expression of uterine activation and inflammatory genes was quantified by qPCR. In Tlr4-/- mice gestation length was increased by ~9 hr, compared to WT controls. This delay in parturition in Tlr4-/- mice was linked to a delay in the expression of uterine activation genes, including prostaglandin receptors, oxytocin receptor and connexion-43. Tlr4-/- mice also showed delayed expression of inflammatory genes Il1b, Il6 and Il12b. Administration of lipopolysaccharide (LPS) to mice to mimic infection-induced preterm labour failed to induce a cytokine response in the female reproductive tract of Tlr4-/- mice, conferring protection from LPS-induced fetal loss and preterm labour. In contrast, WT mice were susceptible to LPS, exhibiting upregulated cytokines and fetal loss. Finally, when TLR4 antagonist was administered to WT mice in late gestation, gestation length was increased by ~16 hr compared to mice given vehicle control. Collectively these data demonstrate that TLR4 plays an important role in the timing of both natural term and infection-induced preterm labour, and that delivery of a TLR4 antagonist may have therapeutic benefits for women at risk of preterm labour.