Delayed puberty may occur in some boys affected with X-linked Spondyloepiphyseal dysplasia tarda (SEDT), which is caused by mutations of the gene encoding a 140 amino acid protein designated Sedlin. Sedlin interacts with the pituitary homeobox 1 (Pitx1) and steroidogenic factor 1 (SF1) transcription factors, which are involved in the development and regulation of the hypothalamic-pituitary-gonadal axis. We have therefore investigated the hypothesis that SEDT associated mutations of Sedlin may disrupt the interaction between Sedlin, Pitx1 and SF1. Full-length Sedlin, Pitx1 and SF1 constructs tagged with cMyc were generated, together with a full-length HA-tagged Sedlin construct into which the SEDT mutations Asp47Tyr, Ser73Leu, Phe83Ser, Val130Asp and Gln131Stop were introduced by site-directed mutagenesis. These constructs were co-transfected into COS7 cells and cell lysates prepared for co-immunoprecipitation assays. These confirmed the interaction between Sedlin, Pitx1 and SF1, but revealed that the SEDT mutations did not disrupt the interactions. However, Sedlin was found to co-immunoprecipitate itself indicating that it may form a homodimer and thereby mask the effects of the SEDT mutations in these assays. The use of native gel electrophoresis demonstrated homodimerization of Sedlin, and the effects of the SEDT mutations were therefore investigated in a yeast two-hybrid system, as yeast does not endogenously express Sedlin. This revealed that all the SEDT mutations, except Asp47Tyr, lead to a loss of interaction with Pitx1 and SF1. Three-dimensional modelling studies of Sedlin revealed that Asp47Tyr resides on the surface whereas all the other mutant residues lie within the hydrophobic core of the protein, and hence are likely to affect the correct folding of Sedlin and thereby disrupt protein-protein interactions. Thus, our studies are the first to demonstrate that Sedlin forms homodimers and that SEDT mutations cause a loss of interaction with Pitx1 and SF1, which may contribute to the delayed puberty associated with SEDT.