Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), and prolonged ER stress leads to cell apoptosis. Despite increasing research in this area, the underlying molecular mechanisms remain unclear. Here, we discover that ER stress upregulates the UPR signaling pathway while downregulating E2F target gene expression and inhibiting the G2/M phase transition. Prolonged ER stress decreases the mRNA levels of E2F2, which specifically regulates the expression of F-Box Protein 5(FBXO5), an F-box protein that functions as an inhibitor of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase complex. Depletion of FBXO5 results in increased ER stress-induced apoptosis and decreased expression of proteins related to PERK/IRE1α/ATF6 signaling. Overexpression of FBXO5 wild-type (not its ΔF-box mutant) alleviates apoptosis and the expression of the C/EBP Homologous Protein (CHOP)/ATF. Mechanistically, we find that FBXO5 directly binds to and promotes the ubiquitin-dependent degradation of RNF183, which acts as a ubiquitin E3 ligase in regulating ER stress-induced apoptosis. Reversal of the apoptosis defects caused by FBXO5 deficiency in colorectal cancer cells can be achieved by knocking down RNF183 in FBXO5-deficient cells. Functionally, we observed significant upregulation of FBXO5 in colon cancer tissues, and its silencing suppresses tumor occurrence in vivo. Therefore, our study highlights the critical role of the FBXO5/RNF183 axis in ER stress regulation and identifies a potential therapeutic target for colon cancer treatment.© 2024. The Author(s).