In this study, effects of heat setting treatment (pre-set heat setting ratio lambda and heat setting temperature T-setting) on the crystalline structures, orientation status, microporous structures, gas permeability, and electrochemical performance of the separators were studied in detail by means of differential scanning calorimetry (DSC), infrared dichroism, scanning electron microscope (SEM), ultrasmall angle X-ray scattering (U-SAXS), Gurley value test, and electrochemical measurement. It was found that with the decrease of lambda or the increase of T-setting, the melting temperature and degree of crystallinity change regularly and the orientation degree decreases gradually, resulting in small-sized micropores and lower porosity and S/V ratio of the separator. Finally, the gas permeability and ionic conductivity of the separator decreased while the dimensional stability increased. By tuning lambda and T-setting, the microstructure, morphology, and performance of the separators can be efficiently controlled.