In this study, nine types of polyacrylonitrile (PAN) nanofibrous membranes with varied fiber diameters and different membrane porosities are prepared by electrospinning followed by hot-pressing. Subsequently, these membranes are explored as Li-ion battery (LIB) separators. The impacts of fiber diameter and membrane porosity on electrolyte uptake, Li+ ion transport through the membrane, electrochemical oxidation potential, and membrane performance as LIB separator (during charge/discharge cycling and rate capability tests of a cathodic half-cell) have been investigated. When compared to commercial Celgard PP separator, hot-pressed electrospun PAN nanofibrous membranes exhibit larger electrolyte uptake, higher thermal stability, wider electrochemical potential window, higher Li+ ion permeability, and better electrochemical performance in LiMn2O4/separator/Li half-cell. The results also indicate that the PAN-based membrane/separator with small fiber diameters of 200–300nm and hot-pressed under high pressure of 20MPa surpasses all other membranes/separators and demonstrates the best performance, leading to the highest discharge capacity (89.5mAhg−1 at C/2 rate) and cycle life (with capacity retention ratio being 97.7%) of the half-cell. In summary, this study has revealed that the hot-pressed electrospun PAN nanofibrous membranes (particularly those consisting of thin nanofibers) are promising as high-performance LIB separators.