β-Galactosidase is one of the most important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant β-galactosidase in Escherichia coli (E. coli). This enzyme differs from its native version (β-GalWT) in that 6 histidine residues have been added to the carboxyl terminus in the primary sequence (β-GalHis), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of β-GalHis with respect to β-GalWT. Although, both enzymes showed a similar catalytic profile as a function of temperature, β-GalHis presented a higher resistance to the thermal inactivation and evidenced greater half-life time compared to β-GalWT. At room temperature, β-GalHis showed a fluorescence spectrum compatible with a partially unstructured protein however, it exhibited a lower tendency to the thermal-induced unfolding with respect to β-GalWT. Analytical ultracentrifugation experiments demonstrated that the population of β-GalHis molecules exhibited a higher proportion of monomers and a lower proportion of tetrameric species with respect to the His-tag free protein. The impairment of tetramerization may would explain the negative effect of the presence of His-tag on the enzymatic activity. In addition, the present results, analyzed in the context of the available literature, suggest that the effect of the His-tag is protein-specific.