We identified a variant of Escherichia coli STb toxin by PCR amplification of clinical isolates obtained from diseased pigs. The variant differed by only one amino acid at position 12 from His to Asn. This change was observed in 23 of the 100 randomly selected enterotoxigenic E. coli (ETEC) isolates tested. There was a positive correlation between the presence of the STa enterotoxin and the STb variant. As the variant represented a high percentage of the ETEC strains tested, we were interested in determining if the single amino acid change results in altered biological characteristics of the toxin. Circular dichroism analysis revealed that the secondary structure of the variant was similar to wildtype and that their thermal stabilities were similar. Surface plasmon resonance showed that the variant and the wildtype toxins possessed similar binding affinities for sulfatide but the variant exhibited a reduced binding capacity. A flow cytometry-based internalization assay showed that the variant toxin is more internalized into epithelial intestinal cells than the wildtype strain. However, this difference was minor. Overall, our results indicate that while wildtype STb and the variant share similar structural properties, modest differences exist in their internalization.