Two new (1-2) and five known (3-7) dihydro-β-agarofuran derivatives were isolated from the roots of Tripterygium wilfordii. The structures of new compounds were elucidated by spectroscopic techniques, such as UV, IR, HRESIMS and NMR. And the structure of compound 1 was confirmed by X-ray crystallographic. Cytotoxic activity assays against four human tumor cell lines (SK-MEL-2, HCC1806, HUH-7, PANC-1) were assessed for compounds 1-7. Compound 2 exhibited pronounced cytotoxicity against SK-MEL-2 cells with an IC50 value of 9.18 μM. Additionally, compound 5 showed significant cytotoxic effects on SK-MEL-2 and HCC1806 cells with IC50 values of 4.59 μM and 8.14 μM, respectively.