Here we report the first application of combined accurate ROE-distance analysis with DFT calculations of NMR chemical shifts to achieve the relative configuration assignment of a marine natural product, conicasterol F, a new polyhydroxylated steroid isolated from the marine sponge Theonella swinhoei. We demonstrate the substantial advantages of this combined approach as a tool for structural studies of natural products, providing a powerful alternative to, or information to underpin, total synthesis when more classical NMR data analysis fails to provide unequivocal results. In this paper, we also describe the isolation and structure elucidation of conicasterol F and its 24-ethyl derivative, theonellasterol I, and their pharmacological evaluation as human nuclear receptor modulators.