Higher-order structures hierarchy of κ-carrageenan in the presence of potassium was reported. Yet, the destiny of κ-carrageenan in higher-order structures in the colon and their impacts on the gut microbiota community remains to be revealed. This study aims to investigate the fermentation behaviors and trace the structural change of higher-order structures of κ-carrageenan during in vitro fermentation. Herein, κ-carrageenan gels were induced in the presence of 10, 40, and 70 mmol/L potassium ions, and a structural-hierarchical transition from secondary to quaternary structures was observed using an AFM and TEM. Results from GPC-MALLS, TLC, IEC, and GC indicated that Single helices were partially fermented by fecal microbiota, while supercoiled helices (tertiary structures) and intertwined helices networks (quaternary structures) were hardly fermented. Tracing the structural change of higher-order κ-carrageenan suggested that the single helices were partially deformed, as shown by the 60% reduction of single helices width, while supercoiled helices were more resistant to fermentation compared to single helices. The super-strands networks were hardly fermented as their width distribution center had been sustained at around 90 nm from 12 to 48 h. In addition, the 16S rRNA gene (V3-V4) amplicon sequencing results indicated that Bifidobacterium longum showed a positive correlation to the structural hierarchy (R = 0.734, p < 0.01). Together, the higher-order structures hierarchy of κ-carrageenan can resist degradation by human gut microbiota.