Gestational diabetes mellitus (GDM) is increasing worldwide and women with a history of GDM are at risk of developing type 2 diabetes which is a risk factor for periodontitis. The aim of this study was to explore the association between the concentrations of matrix metalloproteinase (MMP)-8 and -9 in gingival crevicular fluid (GCF) during early pregnancy with the periodontal diagnosis and the risk of GDM development. A prospective cohort study, including 314 women, enrolled at 11 to 14weeks of pregnancy was conducted. A complete maternal/obstetric and periodontal exam was performed, and GCF samples were obtained for the MMP-8 and -9 determination by Multiplex Elisa Assays. Mann-Whitney test; Spearman's correlation and log-binomial regression model estimated the association between MMPs concentration in GCF and GDM. Fourteen percent of the pregnancies were diagnosed with GDM. An increase in the concentration of MMP-8 and -9 in women with periodontitis stage III and IV compared to periodontitis stage I was observed (99.31ng/mL [IQR: 85.32] versus 71.95ng/mL [IQR: 54.04], and 262.4ng/mL [IQR: 312.55] versus 114.1ng/mL [IQR: 184.94], respectively). Women who developed GDM showed increased concentrations of MMP-8 and -9 in GCF since the beginning of pregnancy (P=0.0381; P=0.0302, respectively). MMP-8 concentration in GCF was associated with GDM (RR: 1.19; P=0.045; CI 95% 1.00 to 1.40; and RR: 1.20; P=0.063; CI 95% 0.99 to 1.45 in the adjusted model). GCF concentrations of MMP-8 and -9 at early of pregnancy are increased in women with severe periodontitis and associated with the GDM development.