To establish an accurate correlation between a separator’s pore structure and the electrochemical performance of a lithium-ion battery (LIB), we fabricate well defined polyethylene (PE) separators on the same production line while maintaining most processing variables, except for composition. Four PE separators having different thicknesses and porosities (16 μm/37%, 16 μm/40%, 16 μm/47%, 22 μm/47%, respectively) are physically and electrochemically evaluated in detail. Although thickness and porosity remain good parameters by which to represent the separators’ characteristics, both the normalized Gurley number and ionic conductance are found to have much stronger relationships with the rate capability.