The expression and localization of FXYD domain containing ion transport regulator 3 (FXYD3), a transmembrane protein that acts as a chloride channel or chloride channel regulator, was analyzed in pancreatic tissues derived from donors and patients suffering from chronic pancreatitis (CP) or pancreatic ductal adenocarcinoma (PDAC) as well as in pancreatic cancer cells using QRT‐PCR, laser‐capture microdissection and microarray analysis, in situ hybridization and immunohistochemistry. FXYD3 antisense expressing T3M4 pancreatic cancer cells were generated and compared to control cells using anchorage‐dependent and independent growth assays, and xenotransplantation into nude mice. FXYD3 mRNA levels were 3.4‐fold increased in PDAC tissues compared to donor specimens (p = 0.006), and 3.9‐fold increased in microdissected cancer cells compared to normal pancreatic ductal cells (p = 0.02). FXYD3 was localized in the tubular complexes and PanIN lesions of both CP and PDAC, as well as in pancreatic cancer cells. Downregulation of FXYD3 by stable antisense transfection increased significantly the doubling time of T3M4 pancreatic cancer cells from 44 ± 2 hr to 55 ± 12 hr (p = 0.02). Nude mice transplanted with antisense transfected cells displayed a significant increase in tumor doubling time from 3.3 days ± 1.0 to 4.3 days ± 0.43 (p = 0.058). Anchorage‐independent growth and sensitivity to 5‐fluorouracil, gemcitabine and cisplatin as well as to MgCl₂ were not dependent on the level of FXYD3 expression. In conclusion, overexpression of FXYD3 in pancreatic cancer may contribute to the proliferative activity of this malignancy. © 2005 Wiley‐Liss, Inc.