The constant voltage cold start of the proton exchange membrane fuel cell (PEMFC) is usually operated at a low start-voltage in order to ensure high heat generation, which can shorten the process of the PEMFC cold start. However, the effect of constant voltage cold start on the durability of PEMFC is still unclear. Thus, in this work, the PEMFC is tested repeatedly at a low start-voltage to simulate its actual operating state in the vehicle. Then, the effect of the PEMFC durability under constant voltage cold start is investigated by polarization curve, cyclic voltammetry, electrochemical impedance spectroscopy, transmission electron microscope, and ion chromatography. After the repeatedly cold start, the output performance of the PEMFC decreases significantly. According to the characterization results, the degradation mechanism of the PEMFC at the constant voltage cold start is demonstrated to be that the PEMFC start-up repeatedly at low start-voltage leads to the decomposition of membrane polymer structure and promotes the crossover of H2. Meanwhile, the PEMFC start-up repeatedly at low start-voltage also leads to the agglomeration of catalysts, which reduces the active area of catalysts and ultimately results in the degradation of fuel cell performance. Above all, this study proves that the durability of PEMFC can be shortened by the constant voltage cold start at 0.1 V, which provides a reference for the development of the PEMFC cold start control strategy.