Shikonin, a natural naphthoquinone, has attracted much attention due to its various biological activities. Two shikonin glucosides, shikonin-1′,8-di- O -β-D-glucopyranoside ( 1 ) and shikonin-1′- O -β-D-glucopyranoside ( 2 ), were biosynthesized through in vitro enzymatic glycosylation and their structures were elucidated using spectroscopic techniques. The water-solubility and stability of compounds 1 and 2 were significantly higher than those of the parent compound. Furthermore, compound 2 showed moderate cytotoxicity against six cancer cell lines, with IC₅₀ values ranging from 36.10 to 67.47 µM. This research indicated that in vitro enzymatic glycosylation of shikonin is an effective strategy to improve it water solubility and chemical stability.