Cordycepin has good antitumor activity, but its clinical application is limited due to the easy deamination of N6 in structure. In this study, a large lipolysis group was introduced at the cordycepin N6 to improve the problem, cordycepin derivatives ( - ) were synthesized, and biological evaluation of compounds was studied. In this study, the vitro antitumor activity of the compounds against MCF7 cells, HepG2 cells and SGC-7901 cells was evaluated by MTT assay. In the results, compound showed the most obvious inhibitory effect on MCF7 cells with an IC value of 27.57 ± 0.52 μM, which was much lower than cordycepin. Compound showed high selectivity between MCF7 and normal MCF-10A cells. Further biological evaluation showed that compound promoted apoptosis and blocked the cell cycle in the G0/G1 phase. Then, Western Blot was used to detect related apoptotic proteins. It was found that Compound could down-regulate the expression of Bcl-2 protein and up-regulate the expression of p53, Bax, Caspase-3 and Caspase-9 proteins. The mitochondrial membrane potential decreased continuously and the positive expression rate decreased. It was speculated that compound induced the apoptosis of MCF7 cells through the mitochondrial pathway.