Three unusual new steroid thioesters, parathiosteroids A-C (1a-3a), were isolated from the 2-propanol extract of the soft coral Paragorgia sp. collected in Madagascar. Their structures, determined by detailed spectroscopic analysis, were confirmed by synthesis and represent the first isolation of natural steroids bearing a C22 thioester in their side chain. These compounds displayed cytotoxicity against a panel of three human tumor cell lines at the micromolar level. The preparation of several analogues revealed structure/activity relationships in this type of steroids, for example, that the XCH2CH2NHCOCH3 moiety (X = S, O, NH) in the side chain is essential for the antiproliferative activity, and a low degree of oxidation in the A-ring results in higher bioactivity. These natural products could be biosynthetic intermediates in the steroid side chain degradation pathway involving activation with CoA and beta-oxidations.