The structure of trisphaerolide A (1), a mildly cytotoxic metabolite isolated from extracts of the marine sponge Erylus trisphaerus collected in Dominica, has been elucidated by detailed analysis of spectroscopic data. Trisphaerolide A (1) has a putative polyketide biogenesis, incorporating a rare variant on the standard pathway, which involves the addition of methyl branches arising from C-2 carbons of acetate units to chain carbons that arise from C-1 of acetate units.