Platinum supported on carbon support (Pt/C) is currently the most common and practicable electrocatalyst for the real application of polymer electrolyte membrane fuel cells (PEMFCs). In this work, it was found that the nature of a reducing agent has noteworthy influence on Pt nanoparticles growth and distribution over acid-treated-Vulcan carbon support (Pt/AT-VC), which was employed to catalyze the oxygen reduction reaction (ORR) for PEMFC. Three distinct reducing agents, i.e., sodium borohydride (BH), sodium citrate (CA), and formaldehyde (FMY), were employed for Pt/AT-VC preparation through the impregnation-reduction approach. The impacts of the reducing agent on Pt nanoparticles size and its distribution over carbon support were scrutinized by X-ray diffraction (XRD) and high-resolution transmission electron microscopy (TEM) techniques. The electrocatalytic performance for ORR was subsequently studied by a three-electrode setup with rotating ring-disc electrode (RRDE) characterization and practical fuel cell operation. The ORR kinetics and mechanism were confirmed from RRDE, and it was well correlated with the durability test and single-cell results. Based on the results, the catalysts’ performances for practical PEMFC can be arranged in the order of Pt/AT-VC (BH) < Pt/AT-VC (CA) < Pt/AT-VC (FMY), implying the significance of selecting the reducing agent for the preparation of Pt/C for PEMFC real application.