A novel macrolide, spongidepsin (1) has been isolated from the Vanuatu marine sponge Spongia sp. The structure of 1, which contains 9-hydroxy-2,4,7-trimethyltetradeca-14-ynoic acid and N-methylphenylalanine residues joined in a 13-membered ring, was elucidated by spectroscopic analysis. Spongidepsin (1) showed cytotoxic activity against J774.A1, WEHI-164 and HEK-293 cancer cell lines with an IC50 in the sub-micromolar range.