The title compound △14,15-anhydro-24-thiocarbonylbufalin(1) was prepared by the reaction of natural product bufalin with Lawesson reagent. The crystal structure of 1, C24H30O2S·C24H30O2S, was determined by single-crystal X-ray diffraction analysis. It belongs to monoclinic, space group C2, with a = 30.9845(2), b = 6.8036(3), c = 22.5791(15) , V = 4241.7(4) 3, Mr = 384.21, Z = 4, Dc = 1.204 g/cm3, μ = 1.463 mm-1, F(000) = 1664, S = 1.064, R = 0.0487 and wR = 0.0645 for 4683 unique reflections, of which 3757 were observed(I > 2■(I)). The asymmetric unit contains two independent molecules(I and Ⅱ), which are closely similar to each other except for the orientation of the lactone ring. Both conformations of I and Ⅱ are in good agreement with the solution structure in methanol as indicated by 1H-NMR analysis. Due to the presence of heavy atom sulfur in the molecules, the final refinement resulted in a small Flack parameter 0.02(3), permitting the assignments of the absolute configuration. In the solid state, intermolecular hydrogen bonds involving thiocarbonyl group in the lactone moiety and the hydroxyl groups in the steroid moiety ester linked adjacent molecules into a three-dimensional network. Compound 1 showed weak inhibition on Na+/K+-ATPase in contrast to the strong inhibitory activity of the parent compound bufalin, suggesting that the carbonyl group in lactone moiety and the hydroxyl group at C-14 play important roles for the inhibition of Na+/K+-ATPase. The title compound △ 14,15-anhydro-24-thiocarbonylbufalin (1) was prepared by the reaction of natural product bufalin with Lawesson reagent. The crystal structure of 1, C24H30O2S · C24H30O2S, was determined by single-crystal X-ray diffraction analysis (3), c = 22.5791 (15) , V = 4241.7 (4) 3, Mr = 384.21, Z = 4, D = 1.204 g / cm3, μ = 1.463 mm-1, F (000) = 1664, S = 1.064, R = 0.0487 and wR = 0.0645 for 4683 unique reflections, of which 3757 were observed (I> Both conformations of I and II are in good agreement with the solution structure in methanol as indicated by 1H-NMR analysis. Due to the presence of heavy atom sulfur in the molecules, the final refinement resulted in a small Flack parameter 0.02 (3), permitting the assignments of the absolute configuration. In the solid state, intermolecular hydrogen bonds involving thiocarbonyl group in the lactone moiety and the hydroxyl groups in the steroid moiety ester linked adjacent molecules into a three-dimensional network. Compound 1 showed weak inhibition on Na + / K + -ATPase in contrast to the strong inhibitory activity of the parent compound bufalin, suggesting that the carbonyl group in lactone moiety and the hydroxyl group at C-14 play important roles for the inhibition of Na + / K + -ATPase.