BACKGROUND In levonorgestrel intrauterine system (LNG-IUS) users, the prevention of sperm penetration through cervical mucus has not been demonstrated. METHODS Subjects were enrolled in an investigator-blinded study to compare quality and sperm penetrability of mid-cycle cervical mucus between LNG-IUS users and hormone-free controls. Cervical mucus was microscopically examined using World Health Organization (WHO) cervical mucus analysis (CMA). CMA score ≥10 of 15 points indicated cervical mucus favoring sperm penetration. Mucus was incubated with sperm using the WHO simplified slide test (SST) and Kremer sperm cervical mucus penetration test (SCMPT). RESULTS Data from 14 LNG-IUS users and 16 controls showed 14% of LNG-IUS users had CMA score ≥10% vs. 69% of controls (p=.004). SST showed no sperm penetration for LNG-IUS users, significantly less than controls (0% vs. 64.3%, p<.001). SCMPT demonstrated no sperm mucus penetration for LNG-IUS users at 2 and 6 h (0% vs. 85% in controls with 2-h score ≥6, p<.001; 6 h 0% vs. 79% in controls, p<.001). CONCLUSIONS Mid-cycle cervical mucus of LNG-IUS users is poor quality and prevents endocervical sperm transport in vitro.