Magnesium (Mg) deficiency is associated with increased risk and malignancy of colorectal cancer (CRC), but the underlying mechanisms remain elusive. In our study, we used genomic, proteomic, and phosphoproteomic data to reveal the effects of low Mg content on genomic instability and tumor metastasis in CRC. Genomic analysis showed that 160 genes co-occurred and had higher mutation frequencies in Low-Mg tumors, including key driver genes such as KMT2C and ERBB3. Unexpectedly, initiation driver genes of CRC, such as TP53 and APC, displayed high mutation frequencies in High-Mg tumors. Additionally, proteomics and phosphoproteomics indicated that the lack of Mg in tumors could activate epithelial-mesenchymal transition (EMT) by modulating inflammation or remodeling the phosphoproteome of cancer cells. Notably, we observed a negative correlation between the level of phosphorylation of DBN1 at S142 (DBN1S142p) and Mg content. Mutation in S142 to D (DBN1S142D) that mimic the effect of phosphorylation upregulated MMP2 and increased cell migration, while MgCl2 treatment reduced DBN1S142p and reversed this phenotype. Mechanistically, our results suggested that Mg2+ weakened the interaction between DBN1 and ACTN4 by reducing DBN1S142p, enhancing the binding of ACTN4 to F-actin and promoting F-actin polymerization, which ultimately reduced MMP2 expression. These findings shed new light on the significance of Mg deficiency in the advancement of CRC and suggest that Mg supplementation could be a promising strategy for prevention and treatment.