Fibrosarcoma cells exhibit low sensitivity to chemotherapy and significant drug resistance, emphasizing the urgent need for effective, low-toxicity therapeutic agents with reliable production methods and novel treatment strategies. Cordycepin (3’-deoxyadenosine) has shown promising therapeutic potential in cancer treatment. In this study, cordycepin was produced using a genetically engineered Pichia pastoris strain cultured in an inorganic salt medium and purified to over 98% purity via macroporous resin chromatography, providing a cost-effective production alternative. The effects of cordycepin on the human fibrosarcoma cell line HT1080 were assessed using microscopic examination, scratch assays, CCK-8 assays, and flow cytometry (Annexin V-FITC/PI staining). The results demonstrated that cordycepin significantly inhibited cell activity at an effective concentration of 100 μmol/L. Key observations included changes in cell morphology, reduced migration, inhibited proliferation, cell cycle arrest at the G0/G1 and G2/M phases, and induction of apoptosis. Network pharmacology analysis identified 31 potential targets of cordycepin in fibrosarcoma, with its effects on Akt1 (protein kinase B) and disruption of protein phosphorylation pathways emerging as key mechanisms underlying its therapeutic efficacy.