We investigated whether cervical Ureaplasma spp. colonization affects the intensity of inflammatory mediators in amniotic fluid retrieved during cesarean delivery in singleton preterm birth. One hundred fifty-three cases in singleton preterm birth with 24-34 weeks' gestation were enrolled. The intensities of seven inflammatory mediators (interleukin (IL)-1β, IL-6, IL-8, IL-10, tumor necrosis factor-α, and matrix metalloproteins (MMP)-8, MMP-9) of amniotic fluid were measured. We tested cervical swab specimens using real-time polymerase chain reaction assays to detect Ureaplasma spp. colonization. Histologic chorioamnionitis (HCA) was diagnosed when acute inflammation was observed in any of the placental tissues. Mean gestational age at delivery and birth weight were 30.9 ± 2.4 weeks and 1567 ± 524 g, respectively. Cervical Ureaplasma spp. colonization was detected 78 cases. The incidence of HCA was 32.3% (43/133). Although the intensities of all inflammatory mediators were significantly different according to presence or absence of HCA, there were no significant differences according to cervical Ureaplasma spp. colonization. In all 43 cases with HCA and 90 cases without HCA, there were no significant differences between cervical Ureaplasma spp. colonization and the intensity of inflammatory mediators. Cervical Ureaplasma spp. colonization did not affect the intensity of inflammatory mediators in the amniotic fluid retrieved during cesarean delivery.