Our objective was to determine the role of toll-like receptor 4 (TLR4) in uterine ischemia/reperfusion (I/R)-induced fetal growth restriction (FGR). Pregnant TLR4-deficient and wild-type mice were subjected to I/R or a sham procedure. Fetal and placental weights were recorded and tissues were collected. Pep-1 (inhibits low-molecular-weight hyaluronan (LMW-HA) binding to TLR4) was used to determine whether LMW-HA-TLR4 interaction has a role in FGR. TLR4-deficient mice exhibited significantly lower baseline fetal weights compared with wild-type mice (P<0.05), along with extensive placental calcification that was not present in wild-type mice. Following I/R, fetal and placental weights were significantly reduced in wild-type (P<0.05) but not in TLR4-deficient mice. However, I/R increased fetal loss (P<0.05) only in TLR4-deficient mice. Corresponding with the reduced fetal weights, uterine myeloperoxidase activity increased in wild-type mice (P<0.001), indicating an inflammatory response, which was absent in TLR4-deficient mice. TLR4 was shown to have a regulatory role for two anti-inflammatory cytokines: interferon-B1 decreased only in wild-type mice (P<0.01) and interleukin-10 increased only in TLR4-deficient mice (P<0.001), in response to I/R. Pep-1 completely prevented I/R-induced FGR (P<0.001), indicating a potential role for the endogenous TLR4 ligand LMW-HA in I/R-induced FGR. In conclusion, uterine I/R in pregnancy produces FGR that is dependent on TLR4 and endogenous ligand(s), including breakdown products of HA. In addition, TLR4 may play a role in preventing pregnancy loss after uterine I/R.