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