Thermal shrinkage is a severe problem for the conventional polyolefin separators. In this work, we report the excellent performance of bacterial cellulose (BC) nanofibrous membranes as separators for lithium (Li) ion batteries. Properties of BC separator including morphology, ionic conductivity, electrochemical stability, thermal stability, mechanical strength and battery charge-discharge performance are characterized and compared to a commercial separator membrane (Celgard (R) 2325). Because of the unique fibrous and cross-linked three-dimensional network structure, BC separator shows excellent dimensional stability up to 180 degrees C, good ionic conductivity and competitive battery performance.