This study evaluated the prebiotic potential of cello-oligosaccharides (COS) produced from birch (Betula pendula), an under-utilised lignocellulosic source from the forestry industry, on growth performance, mucosal immunity, gut microbiota composition, and antioxidant capacity of juvenile rainbow trout (Oncorhynchus mykiss). In a 45-day trial, the fish were fed with diets containing 0%, 0.1%, 0.5% and 1.5% COS, while a diet containing fructo-oligosaccharides (0.5% FOS) was used as a positive control. Fish fed with the 0.5% and 1.5% COS diets showed significantly (P < 0.05) higher abundance of Ruminococcaceae, Bacillaceae and Lactobacillaceae, in the faecal microbiota. The COS diets also induced higher antioxidant capacity in the gut and serum, but there were no treatment effects (P > 0.05) on growth of rainbow trout. Gene expression analysis of the intestine showed significant elevation (P < 0.05) in expression of complement (c3 and c-type lectin) and receptor (tlr2) genes of the innate immune system in COS-fed fish. However, for cytokine and adaptive immune genes, no significant differences (P > 0.05) in gene transcripts were observed between the COS/FOS diets with the control diet. These results suggest that dietary cello-oligosaccharides can be a useful feed supplement for rainbow trout, which can modulate intestinal microbial communities, innate immune response and antioxidant capacity of the host.