Reproductive tract infections have long been hypothesized to increase the risk of uterine fibroids. Few studies have been conducted, even for the common infection genital Chlamydia trachomatis (gCT), and only with self-reported gCT data. Our investigation used micro-immunofluorescence serology for gCT to characterize past exposure. We used cross-sectional enrollment data from a prospective fibroid study carried out in the Detroit, Michigan, area; ultrasound examinations systematically screened for fibroids. Participants were African-American women aged 23-34 years (recruited in 2010-2012). Age-and multivariable-adjusted logistic regression models were used to estimate odds ratios. A total of 1,587 women (94% of participants) had unequivocal gCT serology results; 22% had fibroids. Those who were seropositive for gCT were less likely to have fibroids (age-adjusted odds ratio = 0.68, 95% confidence interval: 0.54, 0.87; multivariable-adjusted odds ratio = 0.80, 95% confidence interval: 0.62, 1.03). Inverse associations were similar across categories of fibroid size, number, and total volume. Participant groups likely to have had multiple or severe infections (multiple serovar groups, more sex partners, clinically diagnosed chlamydia) all showed statistically significantly reduced odds of fibroids. A protective association of gCT with fibroids was unexpected but plausible. gCT infection might increase immune surveillance and eliminate early lesions. Further investigation on the relationship between fibroid development and reproductive tract infections is needed.