Abstract Context Cervical excision is a risk factor for preterm birth. This suggests that the cervix plays an essential role in the maintenance of pregnancy. Objective We investigated the role of the cervix through proteomic analysis of cervicovaginal fluid (CVF) from pregnant women after trachelectomy surgery, the natural model of a lack of cervix. Methods The proteome compositions of CVF in pregnant women after trachelectomy were compared with those in control pregnant women by liquid chromatography–tandem mass spectrometry and label-free relative quantification. MUC5B/AC expression in the human and murine cervices was analyzed by immunohistochemistry. Regulation of MUC5B/AC expression by sex steroids was assessed in primary human cervical epithelial cells. In a pregnant mouse model of ascending infection, Escherichia coli or phosphate-buffered saline was inoculated into the vagina at 16.5 dpc, and the cervices were collected at 17.5 dpc. Results The expression of MUC5B/5AC in cervicovaginal fluid was decreased in pregnant women after trachelectomy concomitant with the anatomical loss of cervical glands. Post-trachelectomy women delivered at term when MUC5B/AC abundance was greater than the mean normalized abundance of the control. MUC5B levels in the cervix were increased during pregnancy in both humans and mice. MUC5B mRNA was increased by addition of estradiol in human cervical epithelial cells, whereas MUC5AC was not. In a pregnant mouse model of ascending infection, E. coli was trapped in the MUC5B/AC-expressing mucin of the cervix, and neutrophils were colocalized there. Conclusion Endocervical MUC5B and MUC5AC may be barriers to ascending pathogens during pregnancy.