Pestaloamides A and B (1 and 2), two novel alkaloids featuring an unprecedented spiro[imidazothiazoledione-alkylidenecyclopentenone] scaffold, were obtained from the cultures of an endophytic fungus Pestalotiopsis sp. HS30 which inhabited the stems of Isodon xerophilus. Their planar structures and absolute configurations were fully determined by extensive spectroscopic analysis and X-ray crystallography. In addition, both compounds 1 and 2 showed the latent tumor immunotherapy activity through markedly promoting the cell surface engagement of NKG2D ligands involving MICA/B and ULBP1 in HCT116 cells. © 2020, Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.