Development of practical lithium (Li) metal batteries (LMBs) remains challenging despite promises of Li metal anodes (LMAs), owing to Li dendrite formation and highly reactive surface nature. Polyolefin separators used in LMBs may undergo severe mechanical and chemical deterioration when contacting with LMAs. To identify the best polyolefin separator for LMBs, this study investigated the separator-deterministic cycling stability of LMBs under practical conditions, and redefined the key influencing factors, including pore structure, mechanical stability, and chemical affinity, using 12 different commercial separators, including polyethylene (PE), polypropylene (PP), and coated separators. At extreme compression triggered by LMA swelling, isotropic stress release by balancing the machine direction and transverse direction tensile strengths was found to be crucial for mitigating cell short-circuiting. Instead of PP separators, a PE separator that possesses a high elastic modulus and a highly connected pore structure can uniformly regulate LMA swelling. The ceramic coating reinforced short-circuiting resistance, while the cycling efficiency degraded rapidly owing to the detrimental interactions between ceramics and LMAs. This study identified the design principle of separators for practical LMBs with respect to mechanical stability and chemical affinity toward LMAs by elucidating the impacts of separator modification on the cycling performance.