Proteome analysis of grafted cotton exposed to low-temperature stress can provide insights into the molecular mechanistic of chilling tolerance in plants. In this study, grafted and non-grafted cotton plants were exposed to chilling stress (10 °C/5 °C) for 7 d. After the stress, rootstock and scion samples were labeled by 8-plex iTRAQ (isobaric Tags for Relative and Absolute Quantification), followed by two-dimensional liquid chromatography separation and tandem mass spectrometry identification. In total, 68 differential proteins were identified that were induced by low-temperature stress and grafting, and these proteins regulate physiological functioning. Under low-temperature stress, in the cotton seedlings, the proteins responded to the MAPK signaling pathway and calcium signaling pathway enhanced, the metabolisms of carbohydrate, lipid, nucleotide, and amino acid had a tendency to intensify, the proteins related to protein folding and degradation were activated, along with the system of antioxidant enzymes to offset cellular oxidative damage. In contrast, chilling stress reduced oxidative phosphorylation, photosynthesis, and carbon fixation. These data indicated that the physiological changes in cotton seedlings comprise a complex biological process, and the ability of plants to resist this stress can be improved after grafting onto a vigorous rootstock, although this was not obvious in the young plants. Further studies of low-temperature stress and/or graft-related differences in proteins could lead to the identification of new genes associated with chilling tolerance in plants. These data provide the basis for further studies on the molecular mechanism of chilling tolerance and the relationship of grafting and chilling tolerance in cotton.