Three new compounds, arneatas A-C (1–3), together with three known compounds (4–6) were isolated from the roots of Arnebia guttata Bunge. The structures were established on the basis of extensive spectroscopic data including NMR and HRESIMS. All the new compounds (1–3) were tested for their cytotoxic activity against two glioma cell lines (U118-MG and U373-MG) in vitro after treatment for 48 h. Compound 1 exhibited moderate cytotoxic activity against U118-MG and U373-MG glioma cell lines, with IC50 values of 10.4 and 17.5 µM, respectively.