Trichodesmium is a suspected toxin-producing nonheterocystous cyanobacteria ubiquitous in tropical, subtropical, and temperate seas. The genus is known for its ability to fix nitrogen and form massive blooms. In oligotrophic seas, it can dominate the biomass and be a major component of oceanic primary production and global nitrogen cycling. Numerous reports suggest Trichodesmium-derived toxins are a cause of death of fish, crabs, and bivalves. Laboratory studies have demonstrated neurotoxic effects in T. thiebautii cell extracts and field reports suggest respiratory distress and contact dermatitis of humans at collection sites. However, Trichodesmium toxins have not been identified and characterized. Here, we report the extraction of a lipophilic toxin from field-collected T. thiebautii using a purification method of several chromatographic techniques, nuclear magnetic resonance (NMR), mass spectroscopy (MS), and Fourier transformed-infrared spectroscopy (FT-IR). Trichotoxin has a molecular formula of C(20)H(27)ClO and a mass of 318 m/z and possesses cytotoxic activity against GH(4)C(1) rat pituitary and Neuro-2a mouse neuroblastoma cells. A detection method using liquid chromatography/mass spectrometry (LC/MS) was developed. This compound is the first reported cytotoxic natural product isolated and fully characterized from a Trichodesmium species.