The commercial applications of lithium-sulfur (Li-S) batteries are limited by decreased cycling performance caused by the shuttle effect of polysulfides, slower kinetic reaction rates, and lithium anode dendrites. This study demonstrates the important role of diatomite (DT) in regulating cycle performance of modified separator for Li-S batteries. A multifunctional separator (Co/NC/DT) with DT as a carrier assembled high loading cobalt nanocluster is prepared. The N-doped carbon is immobilized via Si-N bond between DT and N-doped carbon. The depth profiles of XPS indicate that the ratio of pyrrolic N to pyridinic N of Co/NC/DT reaches 5.31 at the etching of 100 nm versus 0.83 for Co/NC at surface, which suggesting that mainly N species is pyrrolic N after the addition of DT. Theoretical calculations confirm that cobalt nanoclusters grown on pyrrolic N-doped carbon have a lower adsorption energy for active materials compared to that on pyridinic N-doped carbon and can reduce the energy barrier, especially only 0.09 eV for reduction from Li2S6 to Li2S4. In-situ X-ray diffraction analysis confirms Co/NC/DT can effectively promote formation of Li2S. Lithium anode in-situ optical microscopy observation shows the modified separator could reduce lithium dendrites effectively. Li-S battery assembled with the modified separator can reach an initial capacity of 1331.3 mAh g−1 at 0.2 C and cycle 1500 cycles at 1.0 C with a decay of only 0.039% per cycle. Through regulation of DT, Co/NC/DT accelerates the conversion from long-chain sulfur to short-chain sulfur and Li+ transfer rate, resulting in improving cycling performance.