A series of 1,3-alternate conformation thiacalix[4]arenes containing different isomeric aminopyridyl pendent arms have been synthesized. It was found that their self-assembly behaviors and complexation properties strongly depended on the structures of aminopyridyl pendent arms. The crystal structures demonstrate that tetra(meta-aminopyridyl)-thiacalix[4]arene motif is capable of forming intramolecular hydrogen bondings between the sp2 nitrogen donors in the meta position of the aminopyridyl groups and the facing amide N–H of the adjacent aminopyridyl groups, and self-assembles via C-H/O weak hydrogen bondings and C-H/p interaction to generate a double stranded rectilineal networks. By contrast, in the case of tetra(para-aminopyridyl)-thiacalix[4]arene, the presence of para-aminopyridyl units enables the formation of N-H/N strong hydrogen bondings between the individual molecules leading to the solid-state structure with water-bridged double strands. Their complexation properties had been also studied by measurement of the stability constants for their complexation in a range of metal cations and investigation of their binding models via 1H NMR titration and ESI-MS experiments. It was found that the three ligands exhibited high and selective extractability toward Agþ, and their stoichiometry of ligand to Agþ was 1:1, while the meta-aminopyridyl derivative showed the best extraction capacity and possessed the most efficient binding sites.