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Growing evidence indicates that vitamin B (VB) and vitamin D (VD) are associated with the development of obesity. The effects of supplemental dietary VB and VD on the gut microbiota of obese individuals are still largely unknown. In the present study, 997 normoweight (NW) and 773 overweight (OW) samples were screened from the American Gut Project. The microbial α-diversity was not affected by VB, VD or VB combined with VD (VBD) supplementation in the normoweight population or the overweigh population. VD significantly modulated 20 gut microbial metabolic pathways in NW. Moreover, VBD supplementation significantly affected 3 phyla and 3 families and 22 pathways in the OW gut microbiota. In summary, our results highlight that VD and VBD might be necessary for reshaping the gut microbiota in NW and OW, respectively. [ABSTRACT FROM AUTHOR]