Previous studies have suggested that dietary habits and dysbiosis of gut microbiota contributed to obesity development. Vitamin B12 is produced by microbes; however, the relationships between vitamin B12, gut microbiome, and obesity are understudied. We aimed to determine the association between vitamin B12 status and gut microbiota relative to obesity in 92 Saudi Arabian females aged 19–25 years who were obese (n = 44) or normal weight (n = 48). Anthropometric, biochemical data, and dietary data were collected. The microbial communities of stool samples were characterized using the shotgun metagenomic sequencing technique. The relationship between vitamin B12 status and gut microbiota composition was identified using Pearson correlation analysis. A statistically significant difference was found in bacterial α- and β-diversity between the groups relative to median serum vitamin B12 level (404.0 pg/mL) and body weight. In the total participants, dietary vitamin B12 intake was inversely correlated with Bifidobacterium kashiwanohense and Blautia wexlerae species. In obese participants, dietary vitamin B12 intake was inversely correlated with Akkermansia muciniphila species and species from the Verrucomicrobia phylum, whereas it was positively correlated with Bacteroides species. Our findings indicate that the abundance (frequency) and diversity (richness) of gut microbiota are associated with vitamin B12 levels and obesity in young females.