Diketopiperazine indole alkaloids were isolated from hemp seed for the first time. Structures were elucidated with multiple spectra and chiral HPLC analysis techniques. The isolated compounds were constituents of hemp seed rather than that of fungal contamination. This work support the literature prediction of indole alkaloids in hemp seed. Neoechinulin A could promote SIRT1 expression in HEK293 cell lines.