Colorectal cancer (CRC) is a malignancy with high global incidence and mortality rates, posing a serious threat to human health. Despite favorable outcomes following early detection and surgical intervention, the asymptomatic nature of CRC often results in delayed diagnoses, limiting surgical treatment options. Furthermore, effective therapeutic drugs for CRC remain lacking in clinical practice, highlighting an urgent need to identify novel therapeutic targets. In this study, we identified that prolyl 4-hydroxylase subunit alpha 3 (P4HA3) is significantly upregulated in CRC and is associated with poor prognosis in patients. Both in vitro and in vivo experiments demonstrated that knockdown of P4HA3 induces ferroptosis, thereby inhibiting tumor growth. This ferroptosis induction is closely linked to increased lipid peroxidation, and P4HA3 knockdown promotes ferroptosis by upregulating acyl-CoA synthetase long-chain family member 4 (ACSL4), which regulates polyunsaturated fatty acid-containing phospholipids (PUFA-PLs) biosynthesis. Mechanistically, P4HA3 knockdown stabilizes ACSL4 mRNA by downregulating AUF1, an important RNA-binding protein (RBP) that binds to AU-rich elements (AREs) in the ACSL4 mRNA 3' untranslated region (UTR), thereby preventing its degradation. Additionally, given the lack of research on P4HA3 inhibitors, we employed virtual screening and identified Tubuloside A as a potential therapeutic agent. Tubuloside A promotes the ubiquitin-proteasome degradation of P4HA3, exerting anti-CRC effects. In summary, our findings demonstrate that P4HA3 protects CRC cells from ferroptosis by regulating ACSL4 mRNA stability via AUF1, and Tubuloside A serves as a potential P4HA3 degrader, offering a promising therapeutic strategy for CRC treatment.