Nodal and related proteins play central roles in axes formation, mesendoderm induction, neural patterning, and left-right development. However, Xenopus nodal-related 3 (Xnr3) has unique activities in regulating neural induction and convergent extension movements. Xnr3 is distinguished from other transforming growth factor-beta superfamily members by the absence of the seventh conserved cysteine at the C terminus of the protein, and little is known about the molecular mechanism of Xnr3 action. In this study, we report a novel and unique mechanism of action that the mature region of Xenopus tropicalis nodal-related 3 (Xtnr3) functions as a monomer. Comparative analyses between Xtnr3 and Xnr5 revealed regions required for dimerization: (1) a conserved glycine, (2) the seventh cysteine, and (3) a putative alpha-helix located between the third and the fourth cysteines. These results indicate that the mature region of Nodal-related 3 entirely differs from other Nodal-related proteins in its mechanism of action.