Abstract: Gastric cancer is a leading global malignancy with high incidence and mortality rates, highlighting the urgent need for novel therapeutic agents. As a natural product in the traditional Chinese medicine Arnebiae Radix, the anti-gastric cancer effects of β,β-dimethylacrylalkannin (β,β-DAL) have not been reported. This study aimed to evaluate the in vitro anti-gastric cancer effects of β,β-DAL and elucidate its underlying mechanisms. Through CCK-8 assay, colony formation experiment, and EdU staining, we demonstrated that β,β-DAL significantly suppressed the proliferation of human gastric cancer AGS and HGC-27 cells in a dose-dependent manner, with 48-hour IC₅₀ values of approximately 0.28 and 0.15 μmol·L^-1, respectively. RNA sequencing analysis indicated that the anti-gastric cancer effect of β,β-DAL is associated with the regulation of ferroptosis. Flow cytometry analysis revealed that β,β-DAL upregulated the ROS levels in AGS and HGC-27 cells. In addition, β,β-DAL increased the level of lipid peroxidation in AGS and HGC-27 cells, and the ferroptosis inhibitor ferrostatin-1 reduced their sensitivity to β,β-DAL. Western blot analysis showed that β,β-DAL downregulated the protein expression of stearoyl-CoA desaturase 1 (SCD1) and nuclear factor erythroid 2-related factor 2 (Nrf2), as well as inhibited the ratio of SCD1 to acyl-CoA synthetase long-chain family member 4 (ACSL4) in human gastric cancer AGS and HGC-27 cells. In summary, β,β-DAL may exert anti-gastric cancer effects by inducing ferroptosis in human gastric cancer cells through upregulating ROS levels, increasing intracellular lipid peroxidation, and inhibiting the antioxidant SCD1/Nrf2 pathway. This study provides a candidate drug for gastric cancer treatment and lays the groundwork for further research into the anti-gastric cancer effects of β,β-DAL.