Muscle fructose-1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) and inorganic phosphate, regulates glucose homeostasis by controlling the glyconeogenic pathway. FBPase requires divalent cations, such as Mg2+, Mn2+, or Zn2+, for its catalytic activity; however, calcium ions inhibit the muscle isoform of FBPase by interrupting the movement of the catalytic loop. It has been shown that residue E69 in this loop plays a key role in the sensitivity of muscle FBPase towards calcium ions. The study presented here is based on five crystal structures of wild-type human muscle FBPase and its E69Q mutant in complexes with the substrate and product of the enzymatic reaction, namely F1,6BP and F6P. The ligands are bound in the active site of the studied proteins in the same manner and have excellent definition in the electron density maps. In all studied crystals, the homotetrameric enzyme assumes the same cruciform quaternary structure, with the κ angle, which describes the orientation of the upper dimer with respect to the lower dimer, of -85o. This unusual quaternary arrangement of the subunits, characteristic of the R-state of muscle FBPase, is also observed in solution by small-angle X-ray scattering (SAXS).