Gluco‐oligosaccharides (GlcOS) are potential prebiotics that positively modulate beneficial gut commensals like lactobacilli. For the rational design of GlcOS as prebiotics or combined with lactobacilli as synbiotics, it is important to establish the structure requirements of GlcOS and specificity toward lactobacilli. Herein, the utilization of 10 GlcOS with varied degrees of polymerization (DP) and glycosidic linkages by 7 lactobacilli strains (Levilactobacillus brevis ATCC 8287, Limosilactobacillus reuteri ATCC PTA 6475, Lacticaseibacillus rhamnosus ATCC 53103, Lentilactobacillus buchneri ATCC 4005, Limosilactobacillus fermentum FUA 3589, Lactiplantibacillus plantarum WCFS1, and Lactobacillus gasseri ATCC 33323) was studied. L. brevis ATCC 8287 was the only strain that grew on α/β‐(1→4/6) linked disaccharides, whereas other strains showed diverse patterns, dependent on the availability of genes encoding sugar transporters and catabolic enzymes. The effect of DP on GlcOS utilization was strain dependent. β‐(1→4) Linked cello‐oligosaccharides (COS) supported the growth of L. brevis ATCC 8287 and L. plantarum WCFS1, and shorter COS (DP 2–3) were preferentially utilized over longer COS (DP 4–7) (consumption ≥90% vs. 40%–60%). α‐(1→4) Linked maltotriose and maltodextrin (DP 2–11) were effectively utilized by L. brevis ATCC 8287, L. reuteri ATCC 6475, and L. plantarum WCFS1, but not L. fermentum FUA 3589. Growth of L. brevis ATCC 8287 on branched isomalto‐oligosaccharides (DP 2–6) suggested preferential consumption of DP 2–3, but no preference between α‐(1→6) and α‐(1→4) linkages. The knowledge of the structure‐specific GlcOS utilization by different lactobacilli from this study helps the structural rationale of GlcOS for prebiotic development. [ABSTRACT FROM AUTHOR]