α-Glucans produced by glucansucrase enymes hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown, even with the recent elucidation of glucansucrase crystal structures. Guided by the crystal structure from glucansucrase GTF180-ΔN of Lactobacillus reuteri 180 in complex with the acceptor substrate maltose, we identified several residues (D1028 and N1029 from domain A, as well as L938, A978 and L981 from domain B) near subsite +1 that may be critical for linkage specificity determination and investigated these by random site-directed mutagenesis. First, mutants of A978 (to L, P, F or Y) and D1028 (to Y or W) with larger side chains showed reduced degrees of branching, likely due to the steric hindrance by these bulky residues. Second, L938 mutants (except L938F) and D1028 mutants showed altered linkage specificity, mostly with increased (α1→6) linkage synthesis. Third, mutation of L981 and N1029 significantly affected the transglycosylation reaction, indicating their essential roles in acceptor substrate binding. In conclusion, glucansucrase product specificity is determined by an interplay of domain A and B residues surrounding the acceptor substrate binding groove. Residues surrounding the +1 subsite thus are critical for activity and specificity of the GTF180 enzyme, and play different roles in the enzyme function. This study provides novel insights into the structure-function relationships of glucansucrase enzymes and clearly shows the potential of enzyme engineering to produce tailor-made α-glucans.