<jats:title>Abstract</jats:title><jats:p>Cultivation of the fungus <jats:italic>Penicillium</jats:italic> spp. HS‐3, an endophytic fungus from the stems of <jats:italic>Huperzia serrata</jats:italic>, led to the isolation of a new diketopiperazine alkaloid, named as tryhistatin (<jats:bold>1</jats:bold>), together with three known ones, <jats:bold>2</jats:bold>–<jats:bold>4</jats:bold>, all of which share a similar backbone. The structure of <jats:bold>1</jats:bold> was established by detailed interpretation of the 1D‐ and 2D‐NMR spectra, and HR‐ESI‐MS data. Furthermore, 1D‐ and 2D‐NMR experiments were employed to elucidate the structure of <jats:bold>2</jats:bold> which was deduced previously only on the basis of the LC/MS method. In this article, we confirmed the correctness of the previously reported structure and made a complete assignment of the NMR signals of <jats:bold>2</jats:bold> for the first time.