<jats:title>Abstract</jats:title><jats:p>Aspernigerin (<jats:bold>1</jats:bold>), a novel cytotoxic alkaloid consisting of an unprecedented structural framework has been isolated from the extract of a culture of <jats:italic>Aspergillus niger</jats:italic> IFB‐E003, an endophyte in <jats:italic>Cyndon dactylon</jats:italic>. Its structure was elucidated on the basis of comprehensive NMR spectral analysis and confirmed by single‐crystal X‐ray analysis. Aspernigerin (<jats:bold>1</jats:bold>) has been shown to be cytotoxic to the tumor cell lines nasopharynyeal epidermoid KB, cervical carcinoma Hela, and colorectal carcinoma SW1116 with corresponding IC<jats:sub>50</jats:sub> values of 22, 46, and 35 μ<jats:sc>M</jats:sc>, respectively. A feasible total synthetic route for aspernigerin (<jats:bold>1</jats:bold>) has been established for further pharmacological research.