Firstly, a series of Isosteviol derivatives were synthesized and evaluated for FXa inhibitory activity. Among these compounds, the inhibitory activity of compounds 22, 35 and 38 on FXa was better than that of Isosteviol. Secondly, surface plasmon resonance (SPR) assays were performed for selected compounds. Compounds 22, 35, 38 have similar kinetic signatures, and affinity values were at μM level. Thirdly, compounds 22 and 35 displayed moderate-to-high anticoagulation activity and showed similar sensitivity to PT and aPTT. These findings will provide new insight into the exploration of FXa inhibition.