<p>Colorectal cancer (CRC) is an insidious disease, and therapeutic bottlenecks often arise due to drug resistance and tumor heterogeneity. Ferroptosis, a newly discovered mode of cell death in recent years in oncology, may offer a new direction for cancer treatment. Gentianine (GTN), a natural extract, is reported with multiple pharmacological activities. Herein, GTN’s potential for CRC treatments was studied by investigating its regulatory effects on ferroptosis. HCT116 or SW480 cells were treated with GTN for 24&#xa0;h. Declined proportion of EdU positive cells, reduced migration rate, enhanced cell death, increased malondialdehyde (MDA) and ROS production, restrained superoxide dismutase (SOD) activity, increased Fe²⁺ contents, and downregulated GPX4 and SLC7A11 were observed in GTN-treated HCT116 or SW480 cells, along with downregulated AKT2 and HIF1α, diminished total m6A methylation level and AKT2 N6-methyladenosine, and reduced METTL3 level. Moreover, the repressive function of GTN on proliferation and migration, as well as the facilitating effects of GTN on ferroptosis, was abolished by METTL3 overexpression, AKT2 overexpression, DMOG (dimethyloxallyl glycine, a HIF1α activator), or Fer-1 (a ferroptosis inhibitor). HCT116 xenograft model was established in nude mice, followed by GTN administration. The tumor volume growth, tumor weight, and Ki67 expression in tumor tissues were sharply repressed by GTN, along with increased MDA, restrained SOD activity, downregulated GPX4 and SLC7A11, diminished total m6A methylation level and AKT2 N6-methyladenosine, and reduced METTL3 level in tumor tissues. Collectively, our findings demonstrate that GTN restrains CRC progression by inducing ferroptosis through the inhibition of the novel METTL3/AKT2/HIF1α/SLC7A11 axis, highlighting its potential as a promising therapeutic agent for CRC.</p>