Colorectal cancers (CRC) with microsatellite instability (MSI) display unique clinicopathologic features including a mucinous pattern with frequent expression of the secreted mucins MUC2 and MUC5AC. The mechanisms responsible for this altered pattern of expression remain largely unknown. We quantified DNA methylation of mucin genes (MUC2, MUC5AC, MUC4) in colonic cancers and examined the association with clinicopathological characteristics and molecular (MSI, KRAS, BRAF, and TP53 mutations) features. A control cohort was used for validation. We detected frequent hypomethylation of MUC2 and MUC5AC in CRC. MUC2 and MUC5AC hypomethylation was associated with MUC2 and MUC5AC protein expression (p 5 0.004 and p < 0.001, respectively), poor differentiation (p 5 0.001 and p 5 0.007, respectively) and MSI status (p < 0.01 and p < 0.001, respectively). Interestingly, MUC5AC hypomethylation was specific to MSI cancers. Moreover, it was significantly associated with BRAF mutation and CpG island methylator phenotype (p < 0.001 and p < 0.001, respectively). All these results were confirmed in the control cohort. In the multivariate analysis, MUC5AC hypomethylation was a highly predictive biomarker for MSI cancers. MUC5AC demethylation appears to be a hallmark of MSI in CRC. Determination of MUC5AC methylation status may be useful for understanding and predicting the natural history of CRC. Colorectal cancer (CRC) is a major public health problem with over one million new cases diagnosed worldwide every year. 1 Colorectal carcinogenesis results from a multi-step process associated with accumulation of genetic and epigenetic alterations in epithelial cells. 2 CRC can be subdivided into two main groups defined by the genetic pathways involved. The "suppressor" pathway accounts for 70-80% of CRC, characterized by chromosomal instability (CIN) with loss of heterozygosity of tumor suppressor genes and frequent mutations in APC, KRAS and TP53. The "mutator" pathway, on the other hand, accounts for 12-15% of CRC and is characterized by extensive microsatellite instability (MSI) as a result from defective DNA mismatch repair (MMR) system. 2 Most of MSI cancers are sporadic as a result from somatic epige-netic silencing of the MMR gene MLH1. 3 MSI is also a hallmark feature of Lynch syndrome, as the consequence of a germ line mutation in a MMR gene. 4 Sporadic MSI CRC are thought to arise from serrated polyps through the so-called "serrated neoplasia pathway" involving the BRAF V600E mutation and an extensive DNA methylation pattern known as the CpG island methylator phenotype (CIMP), whereas hereditary MSI CRC seem to arise from conventional