Regulating the transformation of sulfur species is the key to improving the electrochemical performance of lithium-sulfur (Li-S) batteries, in particular, to accelerate the reversible conversion between solid phase Li2S2and Li2S. Herein, we introduced Spidroin, which is a main protein in spider silk, as a dual functional separator coating in Li-S batteries to effectively adsorb polysulfides via the sequence of amino acids in its primary structure and regulate Li+flux through the β-sheet of itssecondary structure, thus accelerating the reversible transformation between Li2S2and Li2S. Spidroin-based Li-S cells exhibited an exceptional electrochemical performance with a high specific capacity of744.1mAh g-1at5C and a high areal capacity of7.5mAh cm-2at a low electrolyte-to-sulfur (E/S) ratio of 6 μL mgs-1and a sulfur loading of8.6mgscm-2.