The Gram‐negative, spiral‐shaped bacterium Helicobacter pylori is a common human pathogen that causes chronic inflammation of the human gastric mucosa, leading to peptic ulceration and/or gastric cancer. Here, we analyzed changes in the phosphoproteome of gastric epithelial cells (AGS) upon infection with H. pylori using a combination of SILAC, phosphoprotein enrichment, 2‐DE, and MALDI TOF/TOF‐MS. From a total of 526 spots we identified 391 protein species (143 proteins) and quantified 332 (127 proteins). Nearly, one‐third of the identified proteins (40/143) were associated with the spliceosome or RNA splicing. The abundance of 20 proteins was altered by H. pylori infection, in particular, a number of serine arginine‐rich (SR) proteins involved in the regulation and control of alternative splicing. Importantly, the combined methodologies enabled the detection of infection‐dependent protein species‐specific regulation, suggesting functional modulation of individual protein species. These findings reveal unexpected new insights into the mechanisms of host cell manipulation by H. pylori, which are likely associated with gastric pathologies, including gastric cancer.