This study investigates the inducing effects of jatrorrhizine (JAT), a bioactive compound extracted from Coptidis Rhizoma, on the p53-mediated ferroptosis in human colorectal cancer (CRC) cell lines HT29 and HCT116, specifically through the modulation of spermidine/spermine N1-acetyltransferase 1 (SAT1). Cell viability was assessed via the cell counting kit-8 (CCK-8) assay, while the colony formation assay was employed to determine the proliferative capacity. Cell cycle distribution was analyzed by flow cytometry, and mitochondrial membrane potential was evaluated via JC-1 dye. High-throughput RNA sequencing (RNA-seq) was performed to analyze transcriptomic changes in JAT-treated HT29 and HCT116 cells. Lentiviral transduction was utilized to generate SAT1-overexpressing HCT116 and HT29 cell lines, with transfection efficiency confirmed by qPCR. The levels of intracellular Fe²⁺, reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) were quantified via specific commercial assay kits. The protein and mRNA levels of key ferroptosis-related markers: solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4), tumor protein p53, and SAT1 were determined by Western blot and qPCR, respectively. The results demonstrated that JAT treatment significantly inhibited both cell viability and proliferation, induced cell cycle arrest, and reduced mitochondrial membrane potential in HCT116 and HT29 cells. RNA-seq results revealed that the KEGG pathways most significantly enriched in JAT-treated cells were protein digestion and absorption, arginine and proline metabolism, glycine, serine and threonine metabolism, and ferroptosis. Notably, the mRNA level of SAT1 in the arginine metabolism pathway was significantly upregulated following JAT treatment. Furthermore, JAT treatment increased the levels of Fe²⁺, ROS, and MDA, while markedly decreasing GSH content in a dose-dependent manner. In addition, the treatment downregulated the protein levels of the ferroptosis inhibitors SLC7A11 and GPX4, and upregulated the expression of the pro-ferroptotic proteins ACSL4, SAT1, and p53. SAT1 overexpression inhibited cell proliferation and activated the p53-mediated ferroptosis signaling pathway. The combination of SAT1 overexpression and JAT treatment (oveSAT1+JAT group) exhibited a synergistic effect, showing superior efficacy over SAT1 overexpression alone (oveSAT1 group). In conclusion, JAT activates the p53-dependent ferroptosis signaling pathway by upregulating the key metabolic enzyme SAT1, which is involved in proline metabolism, thereby inhibiting colorectal cancer cell proliferation.