Three new pyridine type alkaloids, (−)-vinmajpyridines A–C (1–3), along with two known alkaloids, have been isolated from the aerial parts of Vinca major cultivated in Pakistan. Their structures have been elucidated by means of NMR and HRESIMS spectroscopic data. The new alkaloids were evaluated for their cytotoxicity against glioma initiating cell lines (GITC-3# and GITC-18#), glioblastoma cell lines (U-87MG and T98G), and lung cancer cell line A-549, but none of them was active at 20 μg/mL concentration. © 2017, The Author(s).