Lithium-sulfur (Li-S) battery with new configuration is demonstrated by inserting a flexible activated carbon nanofiber (ACNF) interlayer between the sulfur cathode and the separator. The ACNF with tunable pore structure is fabricated by a combination of electrospinning polyimide and a subsequent activation treatment. The influence of the textual characteristics of ACNFs on the electrochemical performance of Li-S batteries has been studied. The highly porous ACNF not only effectively intercepts/stabilizes the shuttling migration of polysulfides within the cathode region, but also provides reliable ionic/electronic conductivity for fast kinetics. The lightweight ACNF interlayer with higher specific surface area can yield enhanced cell performance at a low mass ratio of ACNF/sulfur (0.4). An initial specific capacity of 1224mAhg−1 along with high Coulombic efficiency, long cycling stability and good rate capability is achieved in the modified Li-S cell.