The magnetic skyrmions exhibit intriguing topological behaviors, holding promise for future applications in the realm of spintronic devices. Despite recent advancements, achieving spontaneous magnetic skyrmions and topological transitions in magnets featuring uniaxial magnetic anisotropy, particularly at elevated temperatures (>100 K), remains a challenging endeavor. Here, single-crystal Fe5Si3 nanorods with the central symmetry and uniaxial magnetic anisotropy were successfully synthesized on a mica substrate through chemical vapor deposition, which exhibit a high Curie temperature (TC) of about 372 K. The real-time observation, facilitated by Lorentz transmission electron microscopy, revealed the spontaneous formation of magnetic skyrmions and evolution of domains in focused ion beam-prepared Fe5Si3 thin foils. Moreover, Fe5Si3 device transport measurements expose notable magnetoresistance (MR) effects, enabling the interchange between positive and negative MR across specific temperature settings. These results offer various potential avenues for exploring diverse topological spin textures and their formation mechanisms, indicating inventive applications for iron-silicon alloy in the realm of spintronics.