4,6-α-glucosyltransferases (4,6-α-GTs), which converts amylose into α(1-6) bonds-containing α-glucan, possesses great application potential in enzymatic synthesis of dietary fiber. Primers were designed according to the conserved motifs existing in the amino acid sequence of 4,6-α-GTs, and used to amplify a putative GTFB-Like 4,6-α-GTs gene (named as gtf16) from the genomic DNA of Lactobacillus. The gtf16 gene was cloned into the plasmid pET15b, expressed in Escherichia coli BL21(DE3), followed by purification and characterization. The optimum pH and the optimum temperature of the purified enzyme were 5.0 and 40 °C, respectively. The biotransformation product of this enzyme was systematically characterized by thin-layer chromatography, NMR spectroscopy, and hydrolysis reaction. The Gtf16-catalyzed product shows a similar structure to that of the isomalto/malto-polysaccharide (IMMP), which is the amylose-derived product catalyzed by GtfB from Lactobacillus reuteri 121. Moreover, The Gtf16-catalyzed product contains up to 75% of α(1-6) bonds and has an average molecular weight of 23 793 Da. Furthermore, the content of the anti-digestive components was 88.22% upon hydrolysis with digestive enzymes.