Edited by Roger J. Colbran Bacterial pneumonia is a global health challenge that causes up to 2 million deaths each year. Purinergic signaling plays a pivotal role in healthy alveolar epithelium. Here, we used fluorophore-based analysis and live-cell calcium imaging to address the question of whether the bacterial pathogen Streptococcus pneumoniae directly interferes with purinergic signaling in alveolar epithelial cells. Disturbed purinergic signaling might result in pathophysiologic changes like edema formation and atelectasis, which are commonly seen in bacterial pneumonia. Purine receptors are mainly activated by ATP, mediating a cytosolic calcium response. We found that this purinergic receptor P2Y 2-mediated response is suppressed in the presence of S. pneumoniae in A549 and isolated primary alveolar cells in a temperature-dependent manner. Downstream inositol 3-phosphate (IP 3) signaling appeared to be unaffected, as calcium signaling via protease-activated receptor 2 remained unaltered. S. pneumoniae-induced suppression of the P2Y 2-mediated calcium response depended on the P2Y 2 phosphorylation sites Ser-243, Thr-344, and Ser-356, which are involved in receptor desensitization and internalization. Spinning-disk live-cell imaging revealed that S. pneumoniae induces P2Y 2 translocation into the cytosol. In conclusion, our results show that S. pneumoniae directly inhibits purinergic signaling by inducing P2Y 2 phosphorylation and internalization, resulting in the suppression of the calcium response of alveolar epithelial cells to ATP, thereby affecting cellular integrity and function. The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S4.