The glucansucrase GTFA of Lactobacillus reuteri 121 produces an α-glucan (reuteran) with a large amount of alternating (α1 → 4) and (α1 → 6) linkages. The mechanism of alternating linkage formation by this reuteransucrase has remained unclear. GTFO of the probiotic bacterium Lactobacillus reuteri ATCC 55730 shows a high sequence similarity (80%) with GTFA of L. reuteri 121; it also synthesizes an α-glucan with (α1 → 4) and (α1 → 6) linkages, but with a clearly different ratio compared to GTFA. In the present study, we show that residues in loop977 ((970)DGKGYKGA(977)) and helix α4 ((1083)VSLKGA(1088)) are main determinants for the linkage specificity difference between GTFO and GTFA, and hence are important for the synthesis of alternating (α1 → 4) and (α1 → 6) linkages in GTFA. More remote acceptor substrate binding sites (i.e.+3) are also involved in the determination of alternating linkage synthesis, as shown by structural analysis of the oligosaccharides produced using panose and maltotriose as acceptor substrate. Our data show that the amino acid residues at acceptor substrate binding sites (+1, +2, +3…) together form a distinct physicochemical micro-environment that determines the alternating (α1 → 4) and (α1 → 6) linkages synthesis in GTFA.