Obstructions can occur during any stage of colorectal cancer (CRC) and correspond with poor prognosis. Obstructive colorectal cancer (OCRC) is harder and exhibits increased tumor budding and proliferation of myofibroblasts compared to non-obstructive CRC, suggesting that the occurrence of obstruction may be related to extracellular matrix (ECM) remodeling. Here, we found that CRC and OCRC samples differed substantially in ECM composition, specifically in collagen (newly formed and mature) and proteoglycans (including glycosaminoglycan, hyaluronic acid, and chondroitin sulfate). OCRC also exhibited considerable changes in ECM biomechanics and collagen arrangement. Interestingly, OCRC samples presented a notable increase in matrix cancer-associated fibroblasts (mCAFs). The abundance of mCAFs correlated with the accumulation of palmitic acid (PA), and high concentrations of PA increased the secretion of ECM-related proteins by mCAFs. Additionally, PA did not directly affect normal fibroblasts (NFs) but rather activated the NF-κB pathway in tumor cells to stimulate secretion of CSF-1, TGF-β1 and CXCL8, which promoted the activation of NFs into mCAFs and exacerbated ECM stiffening. Drug screening with a natural compound library identified vanillylacetone as a potential inhibitor of PA-induced cytokine secretion and ECM stiffening. These findings highlight intratumoral PA accumulation as a key mechanism driving ECM alterations and OCRC progression and suggest that targeting this axis may be useful for treating CRC patients with risk of obstruction.