Two new alkaloids, nigellisoquinomine (1) with a novel pyrrolo-isoquinoline skeleton, and nigellapyrrolidine (2) possessing a rare glycosylated pyrrolidine moiety, together with two known alkaloids, agrocybenine (3) and 4,6,6-trimethyl-3,4-epoxypiperidin-2-one (4), were isolated from the seeds of Nigella glandulifera Freyn. Their structures were identified by extensive NMR and HR-ESIMS analysis. Compound 1 exhibited potent protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with an IC50 value of 3.65 ± 0.08 μM. © 2016 Phytochemical Society of Europe