Electrochemical impedance spectroscopy was applied for studying copper oxide (CuO) nanowire networks assembled between metallic microelectrodes by dielectrophoresis. The influence of relative humidity (RH) on electrical characteristics of the CuO nanowire-based system was assessed by measurements of the impedanceZ. A slight increase ofZwith increasing RH at low humidity was followed by a three orders of magnitude decrease ofZat RH above 50–60%. The two opposite trends observed across the range of the examined RH of 5–97% can be caused by water chemisorption and physisorption at the nanowire interface, which suppress electronic transport inside the p-type semiconductor nanowire but enhance ionic transport in the water layers adsorbed on the nanowire surface. Possible physicochemical processes at the nanowire surface are discussed in line with equivalent circuit parameters obtained by fitting impedance spectra. The new investigation data can be useful to predict the behavior of nanostructured CuO in humid environments, which is favorable for advancing technology of nanowire-based systems suitable for sensor applications.