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Cell migration is one of the most important processes in which the cytoskeleton plays a main role. The cytoskeleton network is formed by tubulin microtubules, actin filaments, and intermediate filaments (IFs). While the structure and functions of the two aforementioned proteins have been extensively investigated during the last decades, vimentin IFs structure and their role in cell migration and adhesion remain unclear. Here, we investigated polarity determination in rat fibroblasts with either a knocked out vim gene or with a mutation that blocks filament formation on the stage of unit-length filaments (ULFs). Structured illumination microscopy has demonstrated the difference in the morphology of IFs in wild-type fibroblasts and of ULFs in mutant fibroblasts. We have developed an approach to measure cell stiffness separately on the trailing and leading edges using atomic force microscopy. Young's modulus values on the leading and trailing edge of migrating rat fibroblasts differ approximately by two times, being larger on the leading edge. The knockout of the vim gene leads to having comparable values of Young's moduli on both edges. Vimentin-null cells change the direction of migration more frequently than those expressing wild-type or mutated vimentin. Our results have shown the principle role of vimentin, not only in the form of IFs, but also as ULFs, in the determination of the polarity and the directionality of fibroblast migration.