Cervical insufficiency (CI) is associated with intra-amniotic infection or inflammation. Antimicrobial peptides (AMPs) in amniotic fluid may protect the fetus against microbial invasion, giving their broad-spectrum microbiocidal properties. We evaluated changes in amniotic fluid AMP expression in women with CI and assessed whether these changes are related to their pregnancy outcomes. We evaluated amniotic fluid human neutrophil peptide 3 (HNP-3), human β-defensin-2 (hBD-2), and LL-37 levels in 66 women with CI and 25 normal controls at 16-24 weeks of gestation. The CI group was divided into short cervix and cervical dilation groups, and the cervical dilation group was further divided into preterm and full-term delivery groups according to the pregnancy outcomes, and AMP expression was analyzed in each group. HNP-3 and hBD-2 levels were higher in women with CI than in normal controls and in the cervical dilation as compared to the short cervix group. Among women with cervical dilation, 22 delivered at full-term, and 23 had spontaneous preterm births. The hBD-2 level in amniotic fluid mid-pregnancy was higher in the full-term delivery than in the preterm delivery groups. However, LL-37 levels in amniotic fluid were low in women with CI and normal controls. Amniotic fluid HNP-3 and hBD-2 levels increased in women with CI compared with normal controls. Moreover, increased amniotic fluid hBD-2 levels mid-pregnancy were associated with favorable pregnancy outcomes in women with CI. AMPs in the amniotic fluid may participate in host defense against ascending infection in women with CI.