Efficient capture of the greenhouse gas nitrous oxide (N2O) is of great significance for mitigating climate change and resource recovery. In this study, two guanidinium-based hydrogen-bonded organic frameworks (HOFs) with pyridyl nitrogen site isomerism, namely G-5,5'-BPyDC and G-4,4'-BPyDC, were successfully constructed using 2,2'-bipyridine-5,5'-dicarboxylic acid and 2,2'-bipyridine-4,4'-dicarboxylic acid as ligands. The effects of ligand structure on the hydrogen-bonded network, pore environment, and N2O/N2 adsorption and separation performance were systematically investigated by single-crystal X-ray diffraction, thermogravimetric analysis, Hirshfeld surface analysis, and gas adsorption experiments. The results showed that both frameworks were constructed via N-H…O hydrogen bonds. However, the asymmetric unit of G-5,5'-BPyDC contained two methanol molecule, and its free volume and surface area were larger than those of G-4,4'-BPyDC, while the latter exhibited a more compact molecular packing. Both materials exhibited an decomposition temperature above 290℃, showing good thermal stability. Hirshfeld surface analysis revealed that the total contribution of O-H/H-O and N-H/H-N hydrogen bonds in G-5,5'-BPyDC (32.0%) was higher than that in G-4,4'-BPyDC (29.7%). At 25℃ and 4.0 MPa, the N2O adsorption capacity of G-5,5'-BPyDC was 2.32 mmol/g, higher than that of G-4,4'-BPyDC (2.02 mmol/g). IAST calculations indicate that the selectivities of G-5,5'-BPyDC for N2O/N2 (50∶50 and 10∶90) mixtures reached 29.26 and 111.32, respectively, significantly superior to those of G-4,4'-BPyDC (6.61 and 17.03). In conclusion, pyridyl nitrogen site isomerism can effectively optimize N2O/N2 adsorption and separation performance by modulating the pore polarity and hydrogen-bonded network of the frameworks, providing a new strategy for isomer design.