Abstract: This study aimed to investigate the protective mechanism of Cordyceps militaris ferment (YCC) against senescence in skin fibroblasts induced by continuous passage. The activities of catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) content were evaluated to discuss the biological activities of YCC. In order to study the in-depth protective mechanism of YCC on replicative senescence, transcriptome sequencing technology was employed. Differentially expressed genes (DEGs) were screened and further performed Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and KEGG functional enrichment analysis. The protein-protein interaction (PPI) network of DEGs was constructed to find out the hub genes and its regulatory networks by STRING database. The selected IL-17 signaling pathway was further validated by real-time fluorescent quantitative PCR (qRT-PCR). The results showed that YCC significantly enhanced the activity of antioxidant enzymes in replicative senescent cell models (CAT, 37.82 units/mg protein; GSH-Px, 7.76 units/mg protein; and SOD, 50.58 units/mg protein) (P<0.01), while markedly reduced MDA release (approximately 2.49 μmol/mg protein) (P<0.0001). Venn analysis revealed that the sample group could reverse 207 genes that were abnormally expressed in replicative senescence cells. The PPI network analysis selected the top 10 core targets in Betweenness ranking, which were mainly involved in biological processes such as inflammation, apoptosis and oxidative stress regulation. The IL-17 signaling pathway was influenced obviously by DEGs(P<0.01). The qRT-PCR detection results verified that YCC could significantly inhibit the expression of genes related to the IL-17 signaling pathway (IL-17A, IL-17C, Akt1, AP-1, and IL-6) (P<0.05). In conclusion, YCC attenuated inflammation and exerted anti-aging effects by suppressing the IL-17 signaling pathway and subsequent downstream inflammatory factor activation. These findings provide a solid theoretical basis for the application of YCC as a functional food ingredient.