Post-translational modification of proteins namely glycosylation influences cellular behavior, structural properties and interactions including during ovarian follicle development and atresia. However, little is known about protein glycosylation changes occurring in diabetes mellitus in ovarian tissues despite the well-known influence of diabetes on the outcome of successful embryo implantation. In our study, the use of PGC chromatography–ESI mass spectrometry in negative ion mode enabled the identification of 138 N-glycans and 6 O-glycans on the proteins of Streptozotocin-induced (STZ) diabetic mouse ovarian tissues (n = 3). Diabetic mouse ovaries exhibited a relative decrease in sialylation, fucosylation and, to a lesser extent, branched N-linked glycan structures, as well as an increase in oligomannose structures on their proteins, compared with nondiabetic mouse ovaries. Changes in N-glycans occurred in the diabetic liver tissue but were more evident in diabetic ovarian tissue of the same mouse, suggesting an organ-specific effect of diabetes mellitus on protein glycosylation. Although at a very low amount, O-GalNAc glycans of mice ovaries were present as core type 1 and core type 2 glycans; with a relative increase in the NeuGc:NeuAc ratio as the most significant difference between control and diabetic ovarian tissues. STZ-treated mice also showed a trend towards an increase in TNF-α and IL1-B inflammatory cytokines, which have previously been shown to influence protein glycosylation.