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Two-dimensional layered materials have attracted considerable attention since the discovery of graphene. Here we demonstrate that the layered Bi2Sr2Co2O8 (BSCO) can be mechanically exfoliated into single- or few-layer nanosheets. The BSCO nanosheets with four or more layers display bulk metallic characteristics, while the nanosheets with three or fewer layers have a layer-number-dependent semiconducting characteristics. Charge transport in bilayer or trilayer BSCO nanosheets exhibits Mott 2D variable-range-hopping (VRH) conduction throughout 2 K–300 K, while the charge transport in monolayers follows the Mott-VRH law above a crossover temperature of 75 K, and is governed by Efros and Shklovskii-VRH laws below 75 K. Disorder potentials and Coulomb charging both contribute to the transport gap of these nanodevices. Our study reveals a distinct layer number-dependent metal-to-semiconductor transition in a new class of 2D materials, and is of great significance for both fundamental investigations and practical devices.