The mucus filling the human cervical opening blocks the entry to the uterus, but this has to be relative and allow for the sperms to penetrate at ovulation. We have studied this mucus, its content of proteins, mucins, and the mucin O-glycosylation in cervical secretions before, during and after ovulation. Cervical mucosal secretions from twelve subjects were collected, reduced-alkylated, separated with polyacrylamide or agarose/polyacrylamide gel electrophoresis, stained with silver, Alcian blue or Coomassie blue stain. Protein and mucin bands from before and during ovulation were digested and subsequently analyzed by nanoLC-FT-ICR MS and MS/MS. We identified 194 proteins after searches against the NCBI non-redundant protein database and an in-house mucin database. Three gel-forming (MUC5B, MUC5AC, and MUC6) and two transmembrane mucins (MUC16 and MUC1) were identified. For the analysis of mucin O-glycosylation, separated mucins from six individuals were blotted to PVDF-membranes, the O-glycans released by reductive beta-elimination and analyzed with capillary HPLC-MS and MS/MS. At least 50 neutral, sialic acid- and sulfate-containing oligosaccharides were found. An increase of GlcNAc-6GalNAcol core 2 structures and relative decrease of NeuAc residues are typical for ovulation and NeuAc-6GalNAcol and NeuAc-3Gal- epitopes for the non-ovulatory phases. The cervical mucus at ovulation is thus characterized by a relative increase in neutral fucosylated oligosaccharides. This comprehensive characterization of the mucus during the menstrual cycle suggests mucin glycosylation as the major alteration at ovulation, but the relation to the altered physicochemical properties and sperm penetrability is still not understood.