Coibamide A (1) is a new, potent antiproliferative depsipeptide which was isolated from a marine Leptolyngbya cyanobacterium collected from the Coiba National Park, Panama. The planar structure of 1 was elucidated by a combination of NMR spectroscopy and mass spectrometry. Exhaustive 1D and 2D NMR spectroscopy included natural abundance 15N and variable temperature experiments; mass spectrometry included TOF-ESI-MSn and FT-MSn experiments. Chemical degradation followed by chiral HPLC− and GC−MS analyses was used to assign the absolute configuration of 1. This highly methylated cyclized depsipeptide exhibited an unprecedented selectivity profile in the NCI 60 cancer cell line panel and appears to act via a novel mechanism.