The novel ultrathin pre-lithiated V6O13 nanosheets are prepared through a low-temperature exfoliation in argon followed by a secondary hydrothermal lithiation. The smallest electrochemical devices based on nanosheet cathodes are fabricated to in situ probe the intrinsic advantages of pre-lithiated nanosheets. Pre-lithiated nanosheets show one order of magnitude higher in electrical conductivity compared with non-lithiated nanosheets. The conductance of ultrathin pre-lithiated V6O13 nanosheets still remains at the same level after discharge process while that of ultrathin non-lithiated nanosheets decreases over two orders of magnitude. The electrochemical performances of nanosheet cathodes before and after pre-lithiation are compared to further demonstrate the enhancement of the pre-lithiation. Ultrathin pre-lithiated V6O13 nanosheet cathodes exhibit a high discharge capacity of 301 mAh g−1 at the current density of 100 mA g−1. At a higher current density of 1000 mA g−1, the capacity retention after 150 cycles of ultrathin V6O13 nanosheet cathodes is greatly improved from 46% to 98% after pre-lithiation. The enhanced electrical conductance, excellent discharge capacities and cycling performance demonstrate the great potential of ultrathin pre-lithiated V6O13 nanosheets in next-generation high-performance energy storage applications.