Hemp seed, the fruits of non-drug variety of Cannabis sativa L., have been used as food and traditional medicine for thousands of years. Previous phytochemical studies of C. sativa led to the identification of cannabinoids, phenolics, fatty acids, sterols, and lignanamides. In this study, 30 lignanamides were isolated from hemp seed by a series of column chromatographic methods monitored by UPLC-TOF-MS analyses. Their structures were elucidated by NMR and HRMS spectroscopic data, calculated ECD, and CD experimental analyses. Among them, compounds 1 and 2 are novel lignanamides possessing an unique 6/5/5/5 tetracyclic benzo-angular triquinane skeleton. Compound 3 and 4 have unusual spirolignan skeleton featuring with a 1-nitrospiro[4.5]deca-6,9-dien-8-one moiety. Compound 5 possesses an unprecedented neoligan skeleton fused by a caffeic acid and a coumarin group. Subsequent chiral resolution of 1–3 was performed by chiral HPLC separation to afford three pairs of enantiomers (+)- and (–)-1, 2 and 3. Pharmacological targeting of endoplasmic reticulum (ER) stress pathway is emerging as a therapeutic strategy for several neurodegenerative diseases. Pretreatment with compounds 1 and 2 significantly reduced the ER stress-induced neuron cytotoxicity on PC12 cells and SH-SY5Y cells. Our results suggested that lignanamides from the fruits of C. sativa may be developed as a new type of neuroprotective agent.