Potato cultivars are sensitive to frost; thus, freezing damage often results in heavy loss of potato yield. In this study, two wild potato species, Solanum acaule W3, which is frost-resistant and has cold-acclimation ability, and Solanum cardiophyllum Cph12, which is frost-sensitive and cannot be cold-acclimated, were used to research the cell structure and physiological changes that occur during cold acclimation. The results showed that the frost resistance of W3 was enhanced by cold acclimation, while the frost resistance of Cph12 did not change. The subcellular characteristics related to the enhancement of freezing resistance mainly include a decrease in the proportion of the vacuole to total cell volume, integrity of the biomembrane, and orderly arrangement of grana lamellae. At the physiological level, the W3 damage index was correlated with membrane lipid peroxidation system indices (including chlorophyll, malondialdehyde, and the difference between relative conductivity before and after freezing treatment in W3), the activity of the antioxidant enzymes superoxide dismutase and catalase, the contents of the osmotic regulators proline and soluble protein, and the contents of the endogenous hormones salicylic acid (SA), indole acetic acid/abscisic acid (IAA/ABA), and SA/ABA, which indicated that cold acclimation enhanced the freezing resistance of wild potato species W3 by enhancing its original cold-tolerance characteristics. The results could be useful to clarify the cold resistance mechanism of plants, and to provide a theoretical basis for cold-resistance breeding.