Introduction: Ampullary cancer is a relatively rare entity and usually treated by pancreatoduodenectomy followed by adjuvant therapy. The intestinal subtype is associated with markedly improved prognosis after resection. Only few cell lines are available for in vitro studies of ampullary cancer and they have not been collectively characterized. Methods: We characterized available ampullary cancer cell lines by subtype maker expression, epithelial-mesenchymal transition (EMT) features, growth and invasion, drug sensitivity and response to cancer-associated fibroblast conditioned medium (CAF-CM). Results: On the basis of EMT features, subtype marker expression, growth, invasion and drug sensitivity three types of cell lines could be distinguished: mesenchymal-like, pancreatobiliary-like and intestinal-like. In response to CAF-CM, enhanced growth, EMT induction as well as suppression of intestinal differentiation markers were observed, but in a heterogenous pattern. Also proteomic analysis of the CAF response distinguished intestinal-like from other cell lines. Discussion: Most of the available AMPAC cell lines seem to reflect a poorly differentiated pancreatobiliary or mesenchymal-like phenotype, consistent with their origin. We suggest that the best cell line model for intestinal-like AMPAC is the SNU869 cell line, while others seem to reflect aggressive AMPAC subtypes.