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Shiga toxin (Stx) is the key virulence factor in the Shiga Toxin-Producing <i>Escherichia coli</i> (STEC), which can cause diarrhea and hemorrhagic colitis with potential life-threatening complications. There are two major types of Stx: Stx1 and Stx2. Several Stx1/Stx2 subtypes have been identified in <i>E. coli</i>, varying in sequences, toxicity and host specificity. Here, we report a novel Stx2 subtype (designated Stx2m) from three clinical <i>E. coli</i> strains isolated from diarrheal patients and asymptomatic carriers in Sweden and Denmark. The Stx2m toxin was functional and exhibited cytotoxicity in vitro. The two Swedish Stx2m-producing strains belonged to the same serotype O148:H39 and Multilocus Sequencing Typing (MLST) Sequence Type (ST) 5825, while the Danish strain belonged to the O96:H19 serotype and ST99 type. Whole-genome sequencing (WGS) data analysis revealed that the three Stx2m-producing strains harbored additional virulence genes and the macrolide resistance gene <i>mdf</i> (A). Our findings expand the pool of Stx2 subtypes and highlight the clinical significance of emerging STEC variants. Given the clinical relevance of the Stx2m-producing strains, we propose to include Stx2m in epidemiological surveillance of STEC infections and clinical diagnosis.