The in-depth structural analysis of spherulites of artificial cellulose, formed by the enzymatic polymerization of cellobiosyl fluoride, is reported. Both positive- and negative-type spherulites were observed by polarization optical microscopy. Scanning electron microscopy revealed that the negative spherulites have a three-dimensional round shape, where platelike single crystals of the artificial cellulose originate radially from the center. Transmission electron microscopy confirmed that the cellulose chains orient vertically in the platelike crystals, explaining the observation of negative-type spherulites by polarization optical microscopy. Cellulose spherulites can be formed easily via the enzymatic polymerization process, and the occurrence of both positive- and negative-type spherulites is proposed, due to two independent polymerization mechanisms.