Water-soluble, catalytic region of glycosyltransferase B (GTFB/CAT) with good bioactivity, was obtained from Streptococcus mutans UA159 (Ingbritt c). The primers were designed based on the conservative sequences of GTFB gene in S. mutans UA159 (Ingbritt c), as listed in the National Center for Biotechnology Information database and the sequences at the ends of GTFB/CAT. The GTFB/CAT gene was amplified by polymerase chain reaction from S. mutans UA159 (Ingbritt c) and then cloned into vector pET-28b(+) to construct the recombinant pET-28b(+)-GTFB/CAT, which was then transformed into Escherichia coli BL21. Optimal induction conditions were as follows: temperature at 37 ℃ or 30 ℃; time for 4 h; 1 mmol/L of IPTG for induction. The product was purified by Ni2+-NAT resin-affinity chromatography and insoluble inclusion body GTFB/CAT was obtained. Subsequent denaturing and refolding produced the water-soluble protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a clear band at 44 ku, which is consistent with the expected molecular weight, and the purity of protein was approximately 80%. The specific activity of the protein was found to be 1.66 IU/mg by the Somogyi method. The results indicated that water-soluble proteins with biological activity were obtained through the expression of cloned GTFB/CAT gene in Escherichia coli BL21. This will lay a basis on further study of GTF antagonists and their effect as well as mechanism of action in prevention of dental caries.