A series of C21 steroidal glycosides were isolated from the roots and rhizomes of Vincetoxicum atratum (Bunge) C. Morren et Decne., including a new compound, cynatroside B2 (1), and seven known compounds (2-8). Their structures were identified by comprehensive spectroscopic analyses, including NMR and HR-ESI-MS spectral data. The isolated steroids were evaluated for their anti-inflammatory activity against lipopolysaccharide-induced mouse macrophage RAW264.7 cells. Compound 1 displayed a significant inhibitory effect on NO, TNF-α and IL-1β.