4,6-α-glucanotransferase can synthesize a dietary fiber named isomalto-/maltopolysaccharide (IMMP), and this IMMP has properties of supporting probiotic growth and promoting host health because it is rich in α(16) bonds. It is valuable to understand the transglycosylation mechanism of 4,6-α-GT and modify 4,6-α-GT to synthesize IMMP products with controllable proportions of α(16) bonds. In this study, we docked isomaltose into the active pocket of 4,6-α-GT (GtfBΔN) from Limosilactobacillus fermentum NCC 3057 and obtained a substrate-enzyme docking model. This model indicated that three positions, S346, S348, and L399, were closely related to the α(16)-transglycosylation conformation of the acceptor substrate; thus, 16 mutants were constructed to explore the mechanistic rationale. Among these mutants, the S346T/S348T mutation improved the α(16) bond percentage of glycosidic bonds in the product from 72% (GtfBΔN) to 86%, and combinatorial substitutions at positions S346, S348, and L399 modified the α(16) bond percentage between 6% and 86%. We point out that the hydrophobicity of these three positions affect the α(16) proportion of IMMP product through influencing the acceptor substrate to obtain the α(16)-transglycosylation conformation. This research provides insight into the 4,6-α-GT transglycosylation mechanism.