We report on the preparation and characterization of indium tin oxide (ITO) nanofiber films with a patterned architecture that are transparent and conductive with a uniform fiber size. ITO nanofiber films with a crisscross pattern were prepared by the electrospinning of a precursor solution containing ethanol, dimethyl formamide (DMF), indium chloride tetrahydrate, tin chloride pentahydrate and poly(vinyl pyrrolidone) (PVP K90) onto a metal mesh template, followed by calcinations after transfer to a glass substrate. The resulting ITO nanofibers had diameters of the order of 100 nm and were composed of single-crystalline nanoparticles that were pure in chemical composition. The morphology, crystallinity and performance of the resulting nanofibers could be controlled mainly by calcination. Optical and electrical investigations demonstrated that these nanofiber films are transparent conductors with an optical transmittance as high as 92%. The resulting patterned ITO nanofiber films would be suitable for applications such as solar cells, sensors and electromagnetic field filters.