〔Abstract〕 Objective To explore the role of Golgi protein ACBD3 knockdown in ferroptosis. Methods ACBD3 knockdownviashRNA, combined with treatment of ferroptosis inducer RSL3, cell viability, iron levels, reactive oxygen species(ROS) levels, and lipid peroxide levels in different treatment groups were measured to comprehensively analyze the role of ACBD3 in ferroptosis. Results Compared to control cells, the levels of ROS increased in ACBD3 knockdown cells. The total iron content was significantly reduced(P<0.001), while the Fe2+concentration in ACBD3 knockdown cells increased compared to control cells(P<0.001). In the presence of ferroptosis inducer RSL3, compared to control cells, cell viability significantly decreased(P<0.001), and lipid peroxide greatly increased in ACBD3 knockdown cells(P<0.001). Conclusion Golgi protein ACBD3 knockdown in mammalian cells changes iron homeostasis and promotes ferroptosis.