Rocky desertification is a bottleneck that reduces ecological and environmental security in karst areas. Paper mulberry, a unique deciduous tree, shows good performance in rocky desertification areas. Its resistance mechanisms are therefore of high interest. In this study, a lysine acetylation proteomics analysis of paper mulberry seedling leaves was conducted in combination with the purification of acetylated protein by high-precision nano LC-MS/MS. We identified a total of 7130 acetylation sites in 3179 proteins. Analysis of the modified sites showed a predominance of nine motifs. Six positively charged residues: lysine (K), arginine (R), and histidine (H), serine (S), threonine (T), and tyrosine (Y) occurred most frequently at the +1 position, phenylalanine (F) was both detected both upstream and downstream of the acetylated lysines; and the sequence logos showed a strong preference for lysine and arginine around acetylated lysines. Functional annotation revealed that the identified enzymes were mainly involved in translation, transcription, ribosomal structure and biological processes, showing that lysine acetylation can regulate various aspects of primary carbon and nitrogen metabolism and secondary metabolism. Acetylated proteins were enriched in the chloroplast, cytoplasm, and nucleus, and many stress response-related proteins were also discovered to be acetylated, including PAL, HSP70, and ERF. HSP70, an important protein involved in plant abiotic and disease stress responses, was identified in paper mulberry, although it is rarely found in woody plants. This may be further examined in research in other plants and could explain the good adaptation of paper mulberry to the karst environment. However, these hypotheses require further verification. Our data can provide a new starting point for the further analysis of the acetylation function in paper mulberry and other plants.