Porous structure-tuned cellulose nanofiber paper separators (designated as S-CNP separators) are demonstrated as a promising alternative to commercial polyolefin separators for use in lithium-ion batteries. A new architectural strategy based on colloidal silica (SiO2) nanoparticle-assisted structural control is presented to overcome the difficulty in forming controllable porous structure of pure cellulose nanofiber paper separators (designated as CNP separators) from densely-packed cellulose nanofibers (CNFs). The new S-CNP separators proposed herein incorporate SiO2 nanoparticles as a CNF-disassembling agent (i.e., as non-conductive spacer particles). This structural uniqueness allows loose packing of CNFs, thereby facilitating the evolution of more porous structure. The unusual porous structure of S-CNP separators can be fine-tuned by varying SiO2 contents in the CNF suspension. Notably, the S-CNP separator (fabricated with 5 wt.% SiO2 content) exhibits the highest ionic conduction due to the well-balanced combination of nanoporous structure and separator thickness, thus contributing to excellent cell performance. This study underlines that the colloidal SiO2 nanoparticle-directed structural tuning of CNPs offers a promising route for the fabrication of advanced paper separators with optimized attributes and functionality.