Fructose-6-phosphate aldolase B (FSAB) from Escherichia coli was successfully over-expressed as His-tagged recombinant protein. A decameric protein was observed as for FSAA. Unlike FSAA, FSAB is not thermally stable at temperatures higher than 60 °C. The 70% identity between the two aldolases has allowed the generation of a 3D structure which has shown a high similarity of the two active sites. Full kinetic studies towards several substrates have revealed that FSAB catalytic activity is very close to FSAA activity, corroborated by the similarity of their active sites. FSAB has been able to react with three known donors (dihydroxyacetone, hydroxyacetone and glycolaldehyde) but always slightly slower than FSAA.