Protein-based bioliquid crystals integrate the biological properties of proteins with the physical characteristics of liquid crystals (LCs), presenting novel opportunities in materials science and biomedicine. However, developing protein-based LCs from silk fibers is challenging due to the need to preserve their crystalline structure during isolation and to prevent nanofiber aggregation for controlled self-assembly. Herein, a trace alkali-assisted top-down exfoliation approach is developed to bring silk fibers back to colloidal silk liquid crystalline mesogens (SLCMs) with nanofiber structures (40–90 nm in diameter, 300–900 nm in length). The selected trace alkali concentration (0.089 M) maintains the crystalline structure of silk, yielding ∼ 56.5 % SLCMs. The SLCMs display a high aspect ratio (6.74), a high modulus (4.5 GPa), and a negative zeta potential (−44 ± 3.0 mV), promoting suspension fluidity and spontaneous self-assembly into nematic phases in water. After long-time standing (> 7 days), an aqueous dispersion of SLCMs separates into an upper isotropic phase and a lower liquid crystalline phase, with the liquid crystalline volume fraction increasing with concentration (2.0–5.0 wt%), like many types of lyotropic liquid crystalline systems. Notably, this nanofibers-based LCs have a larger size and are different from the original polymeric lyotropic LCs in the silk secretions. Furthermore, the dual sensitivity of SLCMs to electric fields and shear forces allows the composite materials to exhibit programmable macroscopic shapes and microscopic orientations. These results suggest an alternative approach for preparing protein-based anisotropic nanomaterials with controllable self-assembly, with potential applications in tissue engineering, soft photonics, and biosensors.