BACKGROUND: Wnt5a, a noncanonical Wnt ligand, exhibits dual roles in cancer progression, but its tumor-suppressive mechanisms in colorectal cancer (CRC) remain poorly defined. Stromal-derived signals in the tumor microenvironment (TME) are increasingly recognized as critical modulators of CRC behavior, yet their interplay with therapeutic resistance is unclear. METHODS: Using patient-derived CRC organoids (PDOs) and functional assays, we investigated the role of stromal-secreted Wnt5a. Mechanistic studies combined RNA sequencing, pharmacological inhibition, and immunofluorescence to dissect the Wnt5a/TGF-β/NOTUM/OLFM4 axis. Drug sensitivity assays evaluated the synergy between Wnt5a and 5-fluorouracil (5-FU). RESULTS: Wnt5a was predominantly stromal-derived and suppressed CRC organoid growth by activating TGF-β/Smad2 signaling, which upregulated the Wnt inhibitor NOTUM and downregulated the stemness marker OLFM4. RNA-seq revealed NOTUM induction as the key mediator. Combining Wnt5a with 5-FU synergistically enhanced organoid growth inhibition and cell death, reversing 5-FU-driven NOTUM downregulation. CONCLUSIONS: Our study identifies a novel stromal-TME crosstalk mechanism wherein Wnt5a restrains CRC progression via TGF-β/NOTUM/OLFM4 signaling. The combinatorial efficacy of Wnt5a and 5-FU highlights a promising strategy to overcome chemoresistance. These findings emphasize the therapeutic potential of targeting stromal-derived pathways in CRC.