X-ray crystallography is an important method used to confirm the three-dimensional structure of a chemical compound. In this study, the crystal structure of endosulfan sulfate was investigated. Endosulfan sulfate is the major metabolite of the insecticide endosulfan, which is composed of two stereoisomers (α and β). From GC-MS analysis, α-and β-endosulfan each gave a single peak in the endosulfan sample, but only one peak was observed for endosulfan sulfate. Interestingly, in X-ray crystallography, two conformers of endosulfan sulfate (A and B) were observed at a ratio of 2(A):1(B). A heterocyclic seven-membered ring of conformer B assumed a horizontal-chair form, differing from two twisted forms of α-endosulfan while a vertical-chair form was observed for conformer A, showing the very similar structure to β-endosulfan; this difference in conformation is caused by differing bond angles at O(1)-C(8)-C(3) and O(2)-C(9)-C(4). In space packing, two asymmetric units were obtained, and three molecules were aligned in the order of A-A-B conformers in each unit. The total potential energy of A was slightly lower (approximately 4 kcal/mol) than B, possibly resulting in the two molecules of A that exist in a rigid crystal state. However, A and B conformers should not exist at room temperature in a solution state for GC-MS analysis, likely due to the small energy difference. which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.