Abstract: 3-Substituted estradiol-pyrimidine derivatives were synthesized starting from estradiol. Estradiol was reacted with dibromo alkane in the presence of K₂CO₃ to give the corresponding ethers, which were reacted with uracil or thymine, to afford sixteen target compounds. MTT assay was used to evaluate the antitumor activity of the target compounds against human cancer cell lines. The results showed that the target compounds exhibited varying degrees of inhibitory activity against the tumor cell lines. Compounds 5 (the connecting chain length of 2, single-access uracil product), 6 (the connecting chain length of 2, dual access uracil product), 17 (the connecting chain length of 8, single-access uracil product), 8 (the connecting chain length of 2, dual-access thymine product)and 16 (the connecting chain length of 6, dual-access thymine product)showed excellent proliferation inhibiting activity against tumor cell lines MCF-7. Compounds 5 and 6 also exhibited good proliferation inhibiting activity against MDA-MB-231. Compound 17 also possessed good proliferation inhibiting activity against SKOV-3. Compound 13 , with the connecting chain length of 6, uracil single-access product, exhibited the most potent inhibition of proliferation against MGC-803. Overall, 3-substituted estradiol-pyrimidine derivatives exhibited better proliferation inhibiting activity against MCF-7 than other tested tumor cells(MDA-MB-231, SKOV-3, NCI-H460 and MGC-803). 3-Substituted estradiol-uracil derivatives possessed better anti-proliferative activity of tumor cells than 3-substituted estradiol-thymidine derivatives.