The nuclear pore complex (NPC) is a vital regulator of molecular transport between the nucleus and cytoplasm in eukaryotic cells. At the heart of the NPC's function are intrinsically disordered phenylalanineglycine-rich nucleoporins (FG-Nups), which form a dynamic permeability barrier within the central channel. This disordered nature facilitates efficient nucleocytoplasmic transport but also poses significant challenges to its characterization, especially within the nano-confined environment of the NPC. Recent advances in experimental techniques, such as cryo-electron microscopy, atomic force microscopy, fluorescence microscopy, and nuclear magnetic resonance, along with computational modeling, have illuminated the conformational flexibility of FG-Nups, which underpins their functional versatility. This review synthesizes these advancements, emphasizing how disruptions in FG-Nup behavior-caused by mutations or pathological interactions-contribute to diseases such as neurodegenerative disorders, aging-related decline, and viral infections. Despite progress, challenges persist in deciphering FG-Nup dynamics within the crowded and complex cellular environment, especially under pathological conditions. Addressing these gaps is critical for advancing therapeutic strategies targeting NPC dysfunction in disease progression.