Preterm delivery (PTD) is a common obstetric complication and a major cause of prenatal morbidity and mortality. The complex inflammatory signals which initiate preterm labour, and how these relate to physiological events in normal term labour, is poorly understood. Bacterial lipopolysaccharide (LPS) is a pathogen-associated molecular pattern (PAMP) which binds TLR4 to activate inflammation and induce PTD in mice. In this study we investigated the role of TLR4 in initiating inflammatory pathways to mediate the pathological events of LPS-induced preterm labour, as well as in initiating the physiological events of normal term delivery, using mice with genetic deficiency in TLR4 (Tlr4 null mutant, Tlr4-/-) or the Myd88 signalling adaptor (Myd88 null mutant, Myd88-/-). A delay in the time of normal term parturition was observed in Tlr4-/-, but not the MyD88-/- mice, suggesting that TLR4 is essential for on-time labour. A higher weight of pups was seen at 24 h after delivery in Tlr4-/- mice compared to wildtype Balb/c mice. Using RT-PCR, expression of the pro-inflammatory cytokine genes in gestational tissues was quantified. Il1a as well as Ifng mRNA were diminished in the uterus, placenta and fetus of the Tlr4-/- mice, compared to the wildtype Balb/c mice on gestational day (gd17), showing that TLR4 is important in activating inflammatory cytokines for in late gestation prior to term delivery. In addition, administration of LPS on gd17 resulted in a significantly lower rate of preterm delivery in Tlr4-/- or Myd88-/- mice, compared to the wildtype Balb/C mice. This was accompanied by the lower expressions of Il12p40 and Il6 in the uterus and placenta of Tlr4-/- or Myd88-/- mice 4 hours after LPS administration on gd 17, when compared to the wildtype mice. Additionally, uterine activation genes Ptgs2 and Ptgfr were induced in the uterus by LPS in Balb/c but not Tlr4-/- or MyD88-/- mice. These results imply a critical role for TLR4 and MyD88 in protecting against LPS-induced preterm delivery by reducing the activation of inflammatory cytokine genes as well as uterine activation genes and show that TLR4-activated inflammation is also crucial for normal time labour.