Subjects with Down syndrome have a high prevalence of periodontal disease. The aim was to investigate the level of Th1-, Th2- and Th17-related cytokines in the gingival crevicular fluid (GCF) of subjects with Down syndrome. Subjects with Down syndrome (n = 24) and controls (n = 29) with a mean age of 16.4 years were clinically examined with respect to periodontal probing depth (PD) and gingival inflammation in terms of bleeding on probing (BOP%). The controls were matched to subjects with Down syndrome regarding age and gingival inflammation (BOP%). All subjects answered a questionnaire regarding oral hygiene, medical history and socioeconomic background. GCF was collected and the concentration of the cytokines, IFN-γ, TNF-α, IL-1β, IL-4, IL-6, IL-10, IL-12 and IL-17 were determined using Bio-Plex cytokine multiplex assays. The volume of GCF (microliters) was significantly higher in subjects with Down syndrome (P < 0.001) compared with controls. The mean concentrations (picogrammes per millilitre) of IL-1β (P < 0.001), IL-4 (P = 0.002), IL-6 (P = 0.005), IL-10 (P = 0.001), IL-12 (P = 0.003), IFN-γ (P = 0.002), and TNF-α (P = 0.002) in GCF, respectively, were significantly higher in subjects with Down syndrome compared with controls. The regression line of the relationship between IFN-γ and IL-4 in GCF differed significantly (P = 0.006) in subjects with Down syndrome compared to controls. Subjects with Down syndrome demonstrated higher concentration of Th1-, Th2- and Th17-related cytokines with an altered relationship between Th1 cytokine, IFN-γ and Th2 cytokine, IL-4, in volume GCF compared to controls.