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Objective To investigate changes in female genital tract bacterial ecology associated with levonorgestrel intrauterine system (LNG IUS) use. Study Design Study participants were regularly cycling Caucasian women without recent history of pregnancy or exogenous hormone use. Vaginal, cervical and uterine samples were obtained at nine time intervals, from 1 week before to 12 weeks after LNG IUS placement. Replicate paired vaginal and cervical specimens were collected to determine the consistency of specimen collection and processing. Results A total of 406 samples from 11 women were available for analysis after DNA extraction, amplification and species identification. Of the 355 bacterial species or genera detected, Lactobacillus crispatus was most prevalent representing 48.9% of over 6 million total reads. L. crispatus reads of replicate vaginal samples were not significantly different [odds ratio (OR) 0.79, 0.36–1.73]. In addition, L. crispatus reads of vaginal and cervical samples from the same visit were not significantly different (OR 0.69, 0.31–1.51). Compared to sampling visits prior to LNG IUS placement, sampling visits after LNG IUS insertion were more likely to have L. crispatus reads greater than 50% of total reads (OR 2.13, 1.01–4.48). Of the 63 uterine samples demonstrating bacteria, Burkholderia genus proteobacteria, a common environmental contaminant, were most prevalent both before and after LNG IUS insertion accounting for 48.0% of all uterine sample reads. Conclusion The vaginal microbiome changes very little in response to LNG IUS placement. Further study is needed to place this finding in context with clinical outcomes. Implications There do not appear to be any clinically important differences in the vaginal microbiome in response to insertion of the LNG IUS.