Artificial hyaluronic acid has been synthesized in vitro via enzymatic polymerization catalyzed by testicular hyaluronidases, which is the first successful example of the hyaluronic acid synthesis via non-biosynthetic pathways. The novel GlcAβ(1→3)GlcNAc oxazoline derivative was designed and synthesized as a transition state analogue monomer for the hyaluronidase catalysis. The oxazoline monomer was efficiently recognized by the enzymes at pH 7.1 to 9.0 and the polymerization reaction proceeded in a regio- and stereo-selective manner to give rise to artificial hyaluronic acid with molecular weight higher than 15000. These results strongly suggest that the transition state of these testicular hyaluronidases catalysis corresponds to a sugar oxazolinium ion.