Down syndrome (DS) patients usually present B-cell deficiency and immune dysregulation characterized by early occurrence of autoimmune disorders, susceptibility to recurrent respiratory tract infections and predisposition to haematological malignancies. Here, we addressed whether the frequency and function of follicular helper (Tfh) and follicular regulatory (Tfr) T cells are altered in DS. Blood was collected from 24 children with DS, nine of which had autoimmunity. Thirty age- and sex-matched healthy control donors (HD) were included as controls. Blood Tfh cell frequencies in DS children were similar to that observed in age-matched HD but were reduced in absolute number. The proportions of CXCR3+ (Tfh1 and Tfh1/17) subsets were significantly increased in DS children as compared to HD, while that of CXCR3- (Tfh17 and Tfh2) were greatly reduced. The expression of PD-1, a molecule that associates with activation status of Tfh, was similar between DS and HD. However, the proportion of CXCR3+PD-1+ Tfh was increased in the patients, while that of CXCR3-PD-1+ was reduced. While no differences in the percentage of Tfr were seen, the ratio of Tfh1, Tfh1/17 and CXCR3+PD-1+ subsets to Tfr was significantly increased in DS patients. Importantly, plasma CXCL13 levels were significantly elevated in DS as compared to HD and positively associated with the proportion of the CXCR3+PD-1+ Tfh cells. Finally, Tfh isolated from DS patients had the capacity to drive the differentiation of B cells into antibody-producing cells. Our findings suggest that alterations in Tfh phenotype, subset distribution and ratio to Tfr might underlie immune dysregulation in DS.