Intrauterine infection/inflammation complicates 25% to 40% of preterm births (PTB). The human vagina is normally populated by Lactobacillus species, some of which upregulate interleukin 10 (IL-10) output in lipopolysaccharide (LPS)-treated human placental trophoblast cells. We hypothesize that a probiotic strain, L rhamnosus GR-1 exerts its anti-inflammatory effect through activation of the Janus Kinases/Signal Transducers and Activators of Transcription (JAK/STAT) and mitogen-activated protein kinase (MAPK) pathways. Placental trophoblasts from term healthy pregnancies were treated with LPS in the presence or absence of pretreatments with GR-1 supernatant and/or chemical inhibitors of the intracellular signaling pathways. Phosphorylation of STAT3 and p38 was measured by Western Blot analysis, and output of IL-10 was determined by enzyme-linked immunosorbent assay (ELISA). Phosphorylation of STAT-3 and p38 was upregulated by GR-1 supernatant alone or in combination with LPS, while IL-10 output was inhibited by both JAK and p38 inhibitors. These data provide an underlying intracellular mechanism for cytokine regulation in the human placenta by L rhamnosus GR-1 and potential prevention of infection/inflammation-mediated PTB.