Ideal delivery systems must effectively overcome the physiological barrier posed by the gastrointestinal mucus layer to achieve efficient transport and absorption of bioactive compounds. Protein self-assembled fibrils have attracted considerable attention due to their unique physicochemical properties, with their morphological and structural characteristics significantly influencing their permeation behavior within the mucus layer. In this study, four types of β-lactoglobulin amyloid fibrils with distinct morphologies, sizes, and stiffnesses were prepared, and their interactions with intestinal mucus as well as their mucus-penetrating capabilities were systematically evaluated. Results from rheology, dynamic light scattering, turbidity analysis, atomic force microscopy, fluorescence spectroscopy, and quartz crystal microbalance with dissipation monitoring revealed that, although electrostatic repulsion exists between negatively charged fibrils and mucin, pronounced fibril-mucin interactions still occur, predominantly driven by hydrophobic forces. This leads varying degrees of adsorption onto the mucin layer or formation of fibril-mucin complexes. Further investigations using Transwell assays, multiple-particle tracking, and laser scanning confocal microscopy demonstrated that, compared with long fibrils (LF), worm-like short fibrils (SF), and crosslinked sheared fibrils (CLFS), the sheared fibrils (LFS) with smaller particle size and moderate flexibility exhibited stronger penetration ability in intestinal mucus, achieving more effective overcoming of the mucus barrier. Overall, this study provides important theoretical insights for the rational design of protein-based fibrillar carriers with efficient mucus-penetrating capabilities for bioactive substances delivery.