A chemical investigation on an endophytic fungus Penicillium expansum isolated from the medicinal plant Plantago depressa Willd. (Plantaginaceae) afforded one new diketopiperazine-type alkaloid, namely penicimine A (1), as well as two known congeners (2 and 3). Their structures were elucidated by widespread spectroscopic data, and the absolute configurations of 1 and 2 were further confirmed by single-crystal X-ray diffraction analyses. Compound 1 represented the first example of benzyl-containing diketopiperazine-type alkaloid bearing a methyl group attached at C-15 position. Compound 1 showed anti-inflammatory activity against LPS-induced nitric oxide (NO) production in RAW264.7 mouse macrophages with an IC50 value of 25.65 μM. © 2022 Informa UK Limited, trading as Taylor & Francis Group.