Gut bacteria provide a rich source of glycosidases that can recognize and/or hydrolyze glycans for nutrition. Interestingly, some glycosidases have also been found to catalyze transglycosylation reactions in vitro and thus can be used for oligosaccharide synthesis. In this work, six putative and one known exo-α-sialidase genes-three from Bacteroides fragilis NCTC9343, three from Clostridium perfringens ATCC 13124, and one known from Bifidobacterium bifidum JCM1254-were subjected to gene cloning and heterogeneous expression in Escherichia coli The recombinant enzymes were purified, characterized for substrate specificity, and screened for transglycosylation activity. A sialidase, named BfGH33C, from B. fragilis NCTC9343 was found to possess excellent transglycosylation activity for the synthesis of sialylated human milk oligosaccharide. The native BfGH33C was a homodimer with a molecular weight of 113.6 kDa. The Km and kcat values for 4-methylumbelliferyl N-acetyl-α-d-neuraminic acid and sialic acid dimer were determined to be 0.06 mM and 283.2 s-1, and 0.75 mM and 329.6 s-1, respectively. The enzyme was able to transfer sialyl from sialic acid dimer or oligomer to lactose with high efficiency and strict α2-6 regioselectivity. The influences of the initial substrate concentration, pH, temperature, and reaction time on transglycosylation were investigated in detail. Using 40 mM sialic acid dimer (or 40 mg/ml oligomer) and 1 M lactose (pH 6.5) at 50°C for 10 min, BfGH33C could specifically produce 6'-sialyllactose, a dominant sialylated human milk oligosaccharide, at a maximal conversion ratio above 20%. It provides a promising alternative to the current chemical and enzymatic methods for obtaining sialylated oligosaccharides.IMPORTANCE Sialylated human milk oligosaccharides are significantly beneficial to the neonate, as they play important roles in supporting resistance to pathogens, gut maturation, immune function, and brain and cognitive development. Therefore, access to the sialylated oligosaccharides has attracted increasing attention both for the study of saccharide functions and for the development of infant formulas that could mimic the nutritional value of human milk. Nevertheless, nine-carbon sialic acids are rather complicated for the traditional chemical modifications, which require multiple protection and deprotection steps to achieve a specific glycosidic bond. Here, the exo-α-sialidase BfGH33C synthesized 6'-sialyllactose in a simple step with high transglycosylation activity and strict regioselectivity. Additionally, it could utilize oligosialic acid, which was newly prepared in an easy, economical way to reduce the substrate cost, as a glycosyl donor. All the studies laid a foundation for the practical use of BfGH33C in large-scale synthesis of sialylated oligosaccharides in the future.