Abstract Background Weaning involves rapid shifts in the gut microbiota, bowel irregularities, and increased infection risks. This exploratory, hypothesis-generating study examined whether daily supplementation with Bifidobacterium longum subsp. infantis M-63 (M-63) may influence bowel function, the gut environment, and general health during weaning. Methods In a randomized, double-blind, placebo-controlled trial, 100 healthy infants and toddlers (5 months to under 3 years) received M-63 (5 billion CFU) or placebo for eight weeks. Stool habits, fecal microbiota, and short-chain fatty acids (SCFAs) were assessed. Cold-like symptoms and medication use were recorded. Exploratory subgroup analyses considered breastfeeding status, age (<1.5 years), and the presence of siblings. Results M-63 supplementation was associated with more days of normal stools and a trend toward fewer diarrhea episodes. Subgroup analyses revealed that breastfed children showed increased B. infantis abundance. In contrast, non-breastfed children exhibited higher fecal SCFA levels. Children <1.5 years and those with siblings showed trends toward fewer cold-like symptoms and reduced antibiotic use. No intervention-related adverse events occurred. Conclusions This exploratory trial suggests M-63 may be associated with modest improvements in gastrointestinal outcomes and selected systemic health-related measures during weaning. These findings should be interpreted cautiously and require confirmation in adequately powered trials. Impact

Daily administration of B. infantis M-63 was associated with potential improvements in bowel habits among infants.

In breastfed children, supplementation with B. infantis M-63 was associated with an observed increase in the relative abundance of B. infantis , alongside a trend toward greater bifidobacterial diversity.

In non-breastfed children, B. infantis M-63 was associated with modestly elevated levels of fecal metabolites, including short-chain fatty acids.

Exploratory findings suggest that supplementation with B. infantis M-63 during weaning may modulate the gut environment and may be associated with immune-related measures, but confirmation in larger, rigorously designed trials is needed.