Pyruvate kinase is an allosterically regulated protein that catalyses the final step of glycolysis to produce pyruvate. Eshchericia coli type I pyruvate kinase (PK1) is allosterically activated by fructose-1,6-bisphosphate (FBP), but the mechanism of regulation for this key glycolytic enzyme remains unknown. Crystal structures obtained from Saccharomyces cerevisiae and Leishmania mexicana PK isozymes identified the FBP binding site and have provided a model for the mechanism of allosteric regulation. However, given the sequence and substrate divergence between the E. coli PK1 and the aforementioned species’ PKs, we anticipate independent mechanisms of regulation. An accurate characterisation of the regulation mechanism of E. coli type I PK awaits a three-dimensional structure of the enzyme in its active state in complex with the FBP activator. Here, an E. coli type I PK mutant has been crystallised to 2.36 Å in the inactive state, and 2.64 Å in the active state with FBP bound. Comparison of this enzyme in the two distinct states provides the information necessary to elucidate the binding interactions that occur between the enzyme and its allosteric activator. Additionally, we have superimposed the structures to investigate the conformational changes that occur upon transition between the active and inactive states.