K5 lyase A (KflA) is a tailspike protein from the K5A phage that catalyzes the degradation of the capsule polysaccharide of K5 strains of Escherichia coli. The K5 E. coli capsule polysaccharide, also known as heparosan, is composed of the disaccharide repeating unit of [-4)-GlcA-β(1,4)-GlcNAc-α(1-] and therefore identical to the biological precursor of heparin and heparan sulfate (HS). KflA could supplement the heparin lyases for heparin and HS analysis. The first part of this study aimed to clarify ambiguity resulting from the revision of the KflA amino acid sequence in 2010 from that published in 2000. We found that only the expression of the updated sequence gave a soluble active enzyme, which produced heparosan degradation products similar to those of previous studies. Next, we examined the specificity of KflA toward heparosan oligosaccharides of varying sizes, all containing a single N-sulfated glucosamine (GlcNS) residue. The presence of GlcNS in an octasaccharide and a nonasaccharide chain directed cleavage by KflA to a single position at the reducing end of the substrate. However, an N-sulfated decasaccharide exhibited extensive cleavage at the nonreducing end of the chain, illustrating a distinct change in the cleavage pattern of KflA toward substrates of differing sizes. Because KflA is able to cleave a substrate containing isolated GlcNS residues, this enzyme could be used for the analysis of low-sulfate content HS domains.