A new caryolane-type sesquiterpene (1) and its isomer at C-12 (2), as well as seven previously known compounds, including a labdane diterpene, an aromatic derivative, and four steroids, were isolated from the aerial parts of Cirsium souliei. The structures of compounds 1 and 2 were established using spectroscopic analysis including one- and two-dimensional NMR, HRESIMS, and IR results. Their absolute configurations were assigned for the first time by comparison of the experimental results with calculated optical rotations. The cytotoxicities of 1 and 2 against a panel of four human cancer cell lines (HL60, Hela, Hep-G2, and Smmc 7721) were evaluated using the MTT assay. The caryolane-type sesquiterpenes are isolated from the genus Cirsium for the first time.