Vitamin B12 is a critical nutrient for humans as well as microbes. Due to saturable uptake, high dose oral B12 supplements are largely unabsorbed and reach the distal gut where they are available to interact with the microbiota. The aim of this study was to determine if oral B12 supplementation in mice alters 1) the concentration of B12 and related corrinoids in the distal gut, 2) the fecal microbiome, 3) short chain fatty acids (SCFA), and 4) susceptibility to experimental colitis. C57BL/6 mice (up to 24 animals/group) were supplemented with oral 3.94 µg/ml cyanocobalamin (B12), a dose selected to approximate a single 5 mg supplement for a human. Active vitamin B12 (cobalamin), and four B12-analogues ([ADE]CN-Cba, [2Me-ADE]CN-Cba, [2MeS-ADE]CN-Cba, CN-Cbi) were analyzed in cecal and fecal contents using liquid chromatography/mass spectrometry (LC/MS), in parallel with evaluation of fecal microbiota, cecal SCFA, and susceptibility to dextran sodium sulfate (DSS) colitis. At baseline, active B12 was a minor constituent of overall cecal (0.86%) and fecal (0.44%) corrinoid. Oral B12 supplementation increased active B12 at distal sites by >130-fold (cecal B12 increased from 0.08 to 10.60 ng/mg, fecal B12 increased from 0.06 to 7.81 ng/ml) and reduced microbe-derived fecal corrinoid analogues ([ADE]CN-Cba, [2Me-ADE]CN-Cba, [2MeS-ADE]CN-Cba). Oral B12 had no effect on cecal SCFA. Microbial diversity was unaffected by this intervention, however a selective decrease in Bacteroides was observed with B12 treatment. Lastly, no difference in markers of DSS-induced colitis were detected with B12 treatment.