A highly cytotoxic macrolide, superstolide A (1), has been isolated from the deep water marine sponge Neosiphonia superstes, collected off New Caledonia. The gross structure was determined by extensive 2D NMR experiments on the lactone 1 and on its opened-ring-derived methyl esters 2 and 3. The relative stereochemistries of the decaline moiety and of the C22-C26 fragment were determined by a combination of NMR data and acetonide analysis on 2. Absolute stereostructure of the decaline portion of 1 has been determined on the basis of GLC-modified Horeau's methodology applied to 4, whereas the results of the application of the modified Mosher's method to 1 and 3 allowed us to propose for the C22-C26 fragment the 22R, 23R, 24R, 25S, 26R configuration. We also propose the solution conformations of superstolide A (1) based on molecular dynamics and mechanics calculations using NMR-derived constraints. Superstolide A was highly cytotoxic against human bronchopulmonary non-small-cell lung carcinoma NSCLC-N6-L16 cells with IC50 of 0.04 μg/mL, murine leukemia cells expressing resistance toward doxorubicine P388 Dox with IC50 of 0.02 μg/mL, murine leukemia P388 cells with IC50 of 0.003 μg/mL, human nasopharyngeal carcinoma KB cells with IC50 of 0.02 μg/mL, and human colon carcinoma HT29 cells with IC50 of 0.04 μg/mL. The isolation and structural elucidation of this compound are described in this report.