We have identified the nonreceptor tyrosine kinase syk as a marker of differentiation/tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). Syk expression is lost in poorly differentiated PDAC cells in vitro and in situ, and stable reexpression of syk in endogenously syk-negative Panc1 (Panc1/syk) cells retarded their growth in vitro and in vivo and reduced anchorage-independent growth in vitro. Panc1/syk cells exhibited a more differentiated morphology and down-regulated cyclin D1, akt, and CD171, which are overexpressed by Panc1 cells. Loss of PDAC syk expression in culture is due to promoter methylation, and reversal of promoter methylation caused reexpression of syk and concomitant down-regulation of CD171. Moreover, suppression of syk expression in BxPC3 cells caused de novo CD171 expression, consistent with the reciprocal expression of syk and CD171 we observe in situ. Importantly, Panc1/syk cells demonstrated dramatically reduced invasion in vitro. Affymetrix analysis identified statistically significant regulation of >2000 gene products by syk in Panc1 cells. Of these, matrix metalloproteinase-2 (MMP2) and tissue inhibitor of metalloproteinase-2 were down-regulated, suggesting that the MMP2 axis might mediate Panc1/mock invasion. Accordingly, MMP2 inhibition suppressed the in vitro invasion of Panc1/mock cells without effect on Panc1/syk cells. This study demonstrates a prominent role for syk in regulating the differentiation state and invasive phenotype of PDAC cells.