Abstract: To explore the effects of donkey bone collagen on the proliferation, migration and secretion of type I collagen of fibroblasts (NHDFs) and its possible signal pathways, the effects of donkey bone collagen on the proliferation and migration of fibroblasts were studied by CCK-8, BrdU and scratch test, and the differentially expressed genes of donkey bone collagen acting on fibroblasts were analyzed by RNA-seq. The results showed that donkey bone collagen had no toxicity to NHDFs cells, and could promote the growth of NHDFs cells within 0.16~5.0 g/mL. 0.75~1.50 g/mL donkey bone collagen could promote the proliferation of NHDFs cells, and the effect of donkey bone collagen on the proliferation of NHDFs cells showed a dose-dependent relationship. 1.50 g/mL donkey bone collagen could significantly promote the migration of NHDFs cells and the secretion of type I collagen. After donkey bone collagen acted on fibroblasts, 384 differentially expressed genes were screened out, of which 172 genes were up-regulated and 212 genes were down-regulated. The results of GO functional enrichment analysis showed that differentially expressed genes were related to neutrophil chemotaxis, neutrophil migration and granulocyte chemotaxis. Enrichment analysis of KEGG pathway showed that differential genes were involved in important biological pathways such as cytokine-cytokine receptor interaction, chemical carcinogenesis, IL-17 and TNF signaling pathway. It was speculated that donkey bone collagen may promote skin wound healing through IL-17 and TNF-α-mediated CXCL chemokines. It clarified the effect of donkey bone collagen in promoting wound healing, filled the gap in the research of donkey bone collagen in promoting wound healing, and would provide ideas for the high-value transformation of donkey bone collagen.