To study the chemical constituents of the whole herb of Selaginella pulvinata and their antitumor activity. Six compounds were isolated from the 95% ethanol extract of S. pulvinata by silica gel, MCI, Sephadex LH-20 column chromatography, as well as semi-preparative HPLC methods. Their structures were identified by NMR and HR-ESI-MS methods as pulvpeptin B (1), selaginellin G (2), selaginellin P (3), selagibenzophenone A (4), N-(2E)-3- (3,4-dihydrophenyl)prop-N_1′-(4-aminobutyl)-3-pyrrole-formaldehyde (5), and cyclo-(L-Hyp-L-Leu) (6). Among them, compound 1 was a new cyclopeptide, and compounds 5 and 6 were obtained from S. pulvinata for the first time. The antitumor activities of compounds 1-6 was determined by MTT assay. The results showed that compounds 1-5 had various inhibitory effects on the proliferation of human liver cancer cells (HepG2) and human lung cancer cells (A549). Compound 1 exhibited the significant inhibitory activity against HepG2 cells and A549 cells with IC₅₀ values of (10.24 ± 0.33), (9.32 ± 0.81) μmol/L, respectively.