Hypermethylation of runt-related transcription factor 3 (RUNX3) promoter regions occurs in at least 65% of colorectal cancer cell lines. Compound K, the main metabolite of ginseng saponin, induced demethylation of a RUNX3 promoter in HT-29 human colorectal cancer cells, assessed by methylation-specific PCR and the quantitative pyrosequencing analysis. The demethylation of RUNX3 in compound K-treated cells resulted in the re-expression of RUNX3 mRNA, protein and the localization into the nucleus. Demethylation of the RUNX3 gene by compound K occurred via inhibition of the expression and activity of DNA methyltransferase 1 (DNMT1). Compound K also significantly induced RUNX3-mediated expression of Smad4 and Bim. DNMT1 inhibitory activity by compound K was related to extracellular signal-regulated kinase (ERK) inhibition, assessed by siRNA transfection on DNMT1 and ERK. In conclusion, compound K significantly inhibits the growth of colorectal cancer cells by inhibiting DNMT1 and reactivating epigenetically-silenced genes. Ginseng saponin is a potential candidate as DNMT1 inhibitor in the chemoprevention of cancer.