Shiga toxin type 2dact (Stx2dact), an Stx2 variant originally identified from Escherichia coli O91:H21 strain B2F1, displays increased cytotoxicity after activation by elastase present in intestinal mucus. Activation is a result of cleavage of two amino acids from the C-terminal tail of the A2 subunit. In this study, we hypothesized that activation leads to increased binding of toxin to its receptor on host cells both in vitro and in vivo. To test this theory, Stx2dact was treated with elastase or buffer alone and then each toxin was assessed for binding to purified globotriaosylceramide (Gb3) in an enzyme-linked immunosorbent assay, or cells in culture by immunofluorescence, or flow cytometry. Elastase- and buffer-treated Stx2dact were also evaluated for binding to mouse kidney tissue and for relative lethality in mice. We found that activated Stx2dact had a greater capacity to bind purified Gb3, cells in culture, and mouse kidney tissue and was more toxic for mice than was non-activated Stx2dact. Thus, one possible mechanism for the augmented cytotoxicity of Stx2dact after activation is its increased capacity to bind target cells, which, in turn, may cause greater lethality of elastase-treated toxin for mice and enhanced virulence for humans of E. coli strains that express Stx2dact.