Aberrant DNA methylation and microRNA expression play important roles in the pathogenesis of pancreatic cancer. While interrogating differentially methylated CpG islands in pancreatic cancer, we identified two members of miR-200 family, miR-200a and miR-200b, that were hypomethylated and overexpressed in pancreatic cancer. We also identified prevalent hypermethylation and silencing of one of their downstream targets, SIP1 (ZFHX1B, ZEB2), whose protein product suppresses E-cadherin expression and contributes to epithelial mesenchymal transition. In a panel of 23 pancreatic cell lines, we observed a reciprocal correlation between miR-200, SIP1, and E-cadherin expression, with pancreatic cancer–associated fibroblasts showing the opposite expression pattern to most pancreatic cancers. In Panc-1 cells, which express SIP1, have low E-cadherin expression, and do not express miR-200a or miR-200b, treatment with miR-200a and miR-200b downregulated SIP1 mRNA and increased E-cadherin expression. However, most pancreatic cancers express miR-200a and miR-200b, but this expression does not affect SIP1 expression, as the SIP1 promoter is silenced by hypermethylation and in these cancers E-cadherin is generally expressed. Both miR-200a and miR-200b were significantly elevated in the sera of pancreatic cancer and chronic pancreatitis patients compared with healthy controls (P < 0.0001), yielding receiver operating characteristic curve areas of 0.861 and 0.85, respectively. In conclusion, most pancreatic cancers display hypomethylation and overexpression of miR-200a and miR-200b, silencing of SIP1 by promoter methylation, and retention of E-cadherin expression. The elevated serum levels of miR-200a and miR-200b in most patients with pancreatic cancer could have diagnostic utility. Cancer Res; 70(13); 5226–37. ©2010 AACR.