Sialylconjugates on cell surfaces are involved in many biological events such as cellular recognition, signal transduction, and immune response. It has been reported that aberrant sialylation at the nonreducing end of glycoconjugates and overexpression of sialyltransferases (STs) in cells are correlated with the malignance, invasion, and metastasis of tumors. Therefore, inhibitors of STs would provide valuable leads for the discovery of antitumor drugs. On the basis of the transition state of the enzyme-catalyzed sialylation reaction, we proposed that the cyclopentane skeleton in its two puckered conformations might mimic the planar structure of the donor (CMP-Neu5Ac) in the transition state. A series of cyclopentane-containing compounds were designed and synthesized by coupling different cyclopentane α-hydroxyphosphonates with cytidine phosphoramidite. Their inhibitory activities against recombinant human ST6Gal-I were assayed, and a potent inhibitor 48l with a Ki of 0.028 ± 0.006 μM was identified. The results show that the cyclopentanoid-type compounds could become a new type of sialyltransferase inhibitors as biological probes or drug leads.