Chemical investigation of Fritillaria verticillata Willd. led to the isolation of eight novel isosteroidal alkaloids (1-4, 6, 9-11) and four known alkaloids (5, 7-8, 12), including three unprecedented cis-fused D/E (13α, 17α) cevanine alkaloids (1-3), one rarely cis-fused E/F (22α) cevanine alkaloid (6), and one uncommon 5β-jervine-type isosteroidal alkaloid featuring a cis-fused A/B ring moiety (11). In order to establish the structures of 1, the calculated NMR with DP4+ evaluation was applied from the plausible structure candidates. The characteristic proton signals for distinguishing D/E conjunction of cevanine alkaloids were summarized. In addition, some proton and carbon signals α to nitrogen in 6 and 7 are unobservable due to the 14 N nuclear quadrupolar relaxation, based on the NMR experiments in different solvents, calculated NMR method and X-ray technology, their structures were determined. The NMR characteristics of cevanine alkaloids to distinguish orientation of long-pair on nitrogen atom with β-hydroxyl at C-20 were also concluded. The anti-inflammatory effects of compounds 1 and 4 were evaluated in LPS-activated RAW 264.7 macrophages. Compound 4 decreased LPS-induced releases of IL-1β and IL-17α in RAW264.7 cells in a dose-dependent manner. Further mechanistic study revealed that 4 suppressed the phosphorylation of IκBα and p65 subunit to regulate the NF-κB signaling pathway.