The behavior of the Seebeck coefficient in the intermediate regime between atomic scale ballistic conduction and bulk-like diffusive conduction remains unclear. To address this, we have developed a microscale device capable of simultaneously measuring the Seebeck coefficient and electrical conductance of gold nanowires in an adiabatic environment. The nanowires were made in situ by electromigration from lithographically prepared bow-tie electrodes, yielding a wide range of wire thicknesses down to a few hundred atoms. We observed quantization of the Seebeck coefficient, a phenomenon previously observed only at the Ångstrom scale, in relatively thick wires with a thickness of several tens of nanometers. The quantized Seebeck coefficient was proportional to the reciprocal of the electrical conductance with a slope of −47.8 μV/K, indicating that electrons are spatially confined due to the electronic shell structure of the nanowire, similar to the quantization of electrical conductance.