Preterm labor (PTL) is a major cause of newborn death and long-term adverse outcome. Several lines of evidence indicate some chemokines are strongly expressed in gestational tissues during labor or PTL. Our clinical data demonstrated a significant elevation of serum CX3CL1 levels in PTL group. To reveal the pathophysiological roles of the CX3CL1-CX3CR1 axis in PTL, we performed present study using lipopolysaccharide (LPS)-induced PTL model in CX3CR1-deficient (Cx3cr1-/-) mice. When WT mice were received LPS, PTL could be observed in 87.5% (7/8 mice). On the contrary, PTL was suppressed in Cx3cr1-/- mice (12.5%; 1/8 mice). Likewise, immunoneutralization of CX3CL1 significantly prevented LPS-induced PTL in WT mice. Intrauterine macrophage recruitment was remarkably reduced in Cx3cr1-/- mice compared with WT mice. The gene expression of inflammatory cytokines such as Il1b, Il6, Tnfa and Ptgs2 related to uterine contraction was lower in Cx3cr1-/- mice than in WT ones. These inflammatory molecules were produced by macrophages, and exogenous CX3CL1 treatment up-regulated the gene expression of them in peritoneal macrophages. Collectively, these findings indicate that the CX3CL1-CX3CR1 axis can have detrimental roles in PTL through macrophage recruitment and macrophage-derived inflammatory mediators and may be a good molecular target for preventing PTL.