Withering is an important biological process associated with dehydration and cell wall metabolism in postharvest plant organs during curing/processing and storage. However, the withering mechanisms involved in dehydration and cell wall metabolism in postharvest tobacco leaves remain elusive. Here, TMT-based quantitative proteomic analysis in tobacco leaves under different withering conditions was performed. In total, 496 differentially abundant proteins (DAPs) were identified. To elucidate the withering mechanism of tobacco leaves, 28 DAPs associated with cell wall metabolism were screened.