Kittens are born with some maternally derived antibodies (MDA) that, together with maternal gut microbiota, bring some form of early defense against pathogens. However, this protection declines and the rapid changes and challenges the kitten typically encounters can adversely affect their health. Nutrition can modulate the immune system and gut microbiota, offering a boost to protection following weaning. This controlled, randomized, double-blinded study tests a dietary supplement containing nucleotides, oligosaccharides, vitamin E and β-carotene in 50 domestic short-haired kittens. Control kittens were fed standard, dry kitten diet whilst test kittens received the same diet with supplement for 52 weeks. Kittens received routine vaccinations and blood samples were collected throughout the study for standard hematology, serum antibodies and cytokine expression. Rectal fecal samples were analyzed for gut microbiota via 16S rRNA gene sequencing. Supplemented kittens showed significantly improved antibody response to Chlamydia vaccine at various timepoints versus controls, with no consistent effects of supplementation on responses to other vaccines. Several cytokines were significantly influenced by the supplement, which also significantly altered gut microbiota diversity. In conclusion, a dietary supplement was shown to promote immune response and gut microbiota changes in kittens and may help to support a healthy transition into adulthood.