Galactokinase catalyses the phosphorylation of α-d-galactose and some structurally related monosaccharides. The enzyme is of interest due to its potential as a biocatalyst for the production of sugar 1-phosphates and due to its involvement in the inherited metabolic disease type II galactosemia. It has been previously shown that a region (residues 231–245) in human galactokinase often has altered mobility when active site residues are varied. We hypothesised that the reverse may be true and that designing changes to this region might affect the functioning of the active site of the enzyme. Focussing on four residues (Leu-231, Gln-242, Glu-244 and Glu-245) we conducted molecular dynamics simulations to explore the effects of changing these residues to glycine or serine. In most cases the variations resulted in local changes to the 231–245 region and global changes to the root mean squared fluctuation (RMSF) of the protein. The four serine variants were expressed as recombinant proteins. All had altered steady state enzyme kinetic parameters with α-d-galactose as a substrate. However, these changes were generally less than ten-fold in magnitude. Changes were also observed with 2-deoxy-α-d-galactose, α-d-galactosamine and α-d-talose as substrates, including (in some cases) loss of detectable activity, suggesting that these variations can tune the specificity of the enzyme. This study demonstrates that activity and specificity of human galactokinase can be modulated by variations designed to affect active site flexibility. It is likely that this principle can be generalised to other enzymes.