It has been recently found that muscle fructose 1,6-bisphosphatase (FBPase) is actively transported into cells' nuclei. Results of an analysis in silico of muscle FBPase structure gave rise to a hypothesis that sequence (203)KKKGK(207) is responsible for nuclear targeting of the enzyme. To test this, HL-1 cardiomyocytes were transfected with FITC-labeled muscle FBPase constructs, bearing mutations within the putative nuclear localization signal (NLS). Results revealed that integrity of the (203)KKKGK(207) motif is critical to nuclear targeting of muscle FBPase and even a single amino-acid change within this sequence results in significant decrease of nuclear accumulation of the enzyme. Although it has long been recognized as a canonical NLS in theoretical and computational research, to the best of our knowledge this is the first experimental evidence confirming that the KKKGK motif can act as a functional NLS in a protein.