<b><i>Background:</i></b> While there is a proven association of upper genital tract<i> Ureaplasma</i> infection during pregnancy with adverse pregnancy outcome, the effect of vaginal <i>Ureaplasma</i> colonization on preterm delivery has been controversially debated. <b><i>Objectives:</i></b> We hypothesized that women with isolation of vaginal <i>U. parvum</i> but not <i>U. urealyticum</i> are at increased risk for spontaneous preterm birth (SPB) compared to women with negative results. <b><i>Methods:</i></b> A vaginal swab taken between 12 and 14 weeks of gestation was analyzed for the presence of <i>Ureaplasma</i> biovars by PCR in 4,330 pregnant women. <b><i>Results:</i></b> Of the study cohort, 37% were positive for <i>U. parvum</i>, 5.9% for <i>U. urealyticum</i>, and 3.1% for both. The rates of SPB were 10.4% (OR 1.7, 95% CI 1.3, 2.2, <i>p</i> < 0.001) and 8.9% (OR 1.4, 95% CI 0.9, 2.3, <i>p</i> = 0.193) in the groups with isolation of <i>U. parvum </i>and <i>U. urealyticum</i>, respectively, compared to 6.4% in the group with negative PCR results. Multiple logistic regression and interaction analyses showed that vaginal colonization with <i>U. parvum</i> but not <i>U. urealyticum</i> was a statistically significant risk factor for SPB (adjusted OR 1.6, 95% CI 1.2, 2.1, <i>p</i> < 0.001), independent of other risk factors such as bacterial vaginosis and history of SPB. <b><i>Conclusion:</i></b> Our study demonstrates a statistically significant and independent association between first-trimester vaginal colonization with <i>U. parvum</i> and subsequent SPB.