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Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens that cause human diseases ranging from diarrhea to life-threatening complications including hemolytic–uremic syndrome. Virulence of STEC strains and their ability to cause severe diseases are associated with the activity of prophage-encoded Shiga toxins (Stxs). The first objective of this work was to isolate and characterize the Stx2d phage from STEC O80:H2 and to study the transfer of this phage in non-STEC strains. The second objective was to assess the survival of Galleria mellonella larvae inoculated with these transduced strains. Firstly, one bacteriophage isolated from a STEC O80:H2 strain was used to infect six non-STEC strains, resulting in the conversion of three strains. Then, stability assays were performed, showing that this phage was stable in the new STEC strains after three successive subculturing steps, as confirmed by a combination of short and long read genome sequencing approaches. This phage, vB_EcoS_ULI-O80_Stx2d, is resistant to moderate temperature and pH. It belongs to a currently unclassified genus and family within the Caudoviricetes class, shares 98% identity with Stx2_112808 phage and encodes several proteins involved in the lysogenic cycle. The yecE gene was identified at the insertion site. Finally, G. mellonella experiments showed that the transduced strains caused significantly higher mortality rates than the corresponding non-STEC strains. In conclusion, this study showed that stx2d gene from O80:H2 E. coli can be transferred to non-STEC strains and contributes to their virulence.