Separators play a critical role in isolating anodes and cathodes for lithium battery. However, commercialized polyolefin-based separators often suffered from poor electrolyte wettability, inferior dimentionally thermal stability, especially the low Young's modulus of polyolefin-based separators cannot effectively inhibit the growth of lithium dendrites, which limits their large-scale practical application in lithium metal batteries. Therefore, a novel separator with high Young's modulus which can be applied to lithium metal batteries is urgently needed. Bacterial cellulose is a porous, nanoscale biopolymer, which is synthesized mainly by micro-organisms such as bacillus xylosus. Compared with plant-derived cellulose, bacterial cellulose exhibits high crystallinity, high thermal stability, high electrolyte absorption, high porosity, and excellent mechanical strength. Considering the above advantages, bacterial cellulose-based bacterial cellulose separators have the potential to be effectively suited in lithium metal batteries. Herein, we demonstrate a novel kind of bacterial cellulose separator. Firstly, the structure of the prepared bacterial cellulose separator is characterized using scanning electron microscopy (SEM), atomic force microspocy (AFM) and X-ray diffraction (XRD), which shows a three-dimensional interpenetrating network structure, suggesting that it is favourable for achieving wetting of the electrolyte and providing more ionic conduction pathways. In addition, this bacterial cellulose separator with high Young's modulus, excellent dimentionally thermal stability and outstanding electrolyte wettability was prepared by a sample process. It is demonstrated that the resultant separator exhibits excellent mechanical strength (144 MPa), high Young's modulus (8.1 GPa) and superior thermal dimentionally stability (≥300 ℃). Meanwhile, Li/Li cells using this bacterial cellulose separator and conventional organic carbonate-based electrolyte can achieve a steady lithium plating/stripping behavior over 4000 h at a current rate of 0.52 mA•cm⁻² and 1.56 mAh•cm⁻². Such improved reliability is chiefly attributed to the high Young's modulus of bacterial cellulose separator and the formed stable solid electrolyte interphase (SEI) which rich in inorganic components such as boron oxides, LiF and Li₂O. More intriguingly, this separator delivers an excellent cycling performance in LiFePO₄/Li battery, stable cycling for 100 cycles with a low capacity loss at 1 C. These fascinating characteristics indicate this separator is a promising material for high-energy-density lithium metal batteries.