A bioassay-guided fractionation of the $CH_2Cl_2$ extract of Selaginella tamariscina yielded six sterols 1-6 such as (4${alpha}$, 5${alpha}$)-4, 14-dimethylcholest-8-en-3-one (1), ergosta-4, 6, 8(14), 22-tetraene-3-one (2), ergosterol endoperoxide (3), 7${eta}$-hydroxycholesterol (4), 7${eta}$-hydroxysitosterol (5), and 7${alpha}$-hydroxysitosterol (6). The structures of isolated compounds were determined using spectroscopic methods. Among these isolates, compounds 2-5 showed potent cytotoxicity against five human tumor cells, while compounds 1 and 6 did not. In the case of compounds 1 and 2, 3-oxo sterol derivatives, compound 1 was inactive, but compound 2 showed potent cytotoxicity. In addition, compound 5 exhibited potent cytotxicity, but, compound 6 which is the 7-epimer of compound 5 was weakly active against tumor cell lines. Therefore, in the case of oxysterol derivatives, the cytotoxicity appeared to be affected by the structural differences, i.e. the configuration of hydroxyl group and the number of conjugated double bond. Taken all together, the present study isolated six sterols from S. tamariscina for the first time based on a bioassay-guided fractionation and indicated that isolated oxysterols could exhibit the cytotoxic effects against tumor cells, suggesting that S. tamariscina might be a promising candidate for the development of anticancer agents.