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Autoinducer‐2 (AI‐2) a signal produced by a range of phylogenetically distant microorganisms, enables inter‐species cell–cell communication and regulates many bacterial phenotypes. Certain bacteria can interfere with AI‐2‐regulated behaviours of neighbouring species by internalizing AI‐2 using the Lsr transport system (encoded by the lsr operon). AI‐2 imported by the Lsr is phosphorylated by the LsrK kinase and AI‐2‐phosphate is the inducer of the lsr operon. Here we show that in Escherichia coli the phosphoenolpyruvate phosphotransferase system (PTS) is required for Lsr activation and is essential for AI‐2 internalization. Although the phosphorylation state of Enzyme I of PTS is important for this regulation, LsrK is necessary for the phosphorylation of AI‐2, indicating that AI‐2 is not phosphorylated by PTS. Our results suggest that AI‐2 internalization is initiated by a PTS‐dependent mechanism, which …