Detergent-resistant membranes of eukaryotic cells are enriched in many important cellular signalling molecules and frequently targeted by bacterial pathogens. To learn more about pathogenic mechanisms of Helicobacter pylori and to elucidate novel effects on host epithelial cells, we investigated how bacterial co-cultivation changes the protein composition of detergent-resistant membranes of gastric adenocarcinoma (AGS) tissue culture cells. Using iTRAQ (isobaric tags for relative and absolute quantification) analysis we identified several cellular proteins, which are potentially related to H. pylori virulence. One of the proteins, which showed a significant infection-dependent increase in detergent resistance, was the polarity-associated serine/threonine kinase MARK2 (EMK1/Par-1b). We demonstrate that H. pylori causes the recruitment of MARK2 from the cytosol to the plasma membrane, where it colocalizes with the bacteria and interacts with CagA. Using Mardin Darby Canine Kidney (MDCK) monolayers and a three-dimensional MDCK tissue culture model we showed that association of CagA with MARK2 not only causes disruption of apical junctions, but also inhibition of tubulogenesis and cell differentiation.