Carotenoids, known for their immunomodulatory and gut microbiota-modulating effects, have drawn attention as potential dietary adjuvants to enhance vaccine efficacy and maintain gut health. This study aimed to evaluate the effect of carotene supplementation on immune response with insight into gut microbiome using an in vivo animal model. The BALB/c mice were fed daily with CaroGaia (50 mg/kg of body weight), a carotene supplement that contained 33.3% α-carotene and 66.6% β-carotene, by oral gavage for 70 days. Mice fed with the vehicle served as controls. The mice in the vaccinated groups received two doses of the CoronaVac inactivated virus vaccine on days 14 and 42. Flow cytometry revealed no significant modulation of lymphocyte subsets (total T lymphocytes, T-helper cells, cytotoxic T lymphocytes, and B cells) with carotene supplementation. In addition, there were no significant differences in the levels of SARS-CoV-2 immunoglobulin G and interferon-gamma in plasma between treatment and control groups. In contrast, the vaccinated carotene group showed an increased SARS-CoV-2 antigen-specific splenocyte proliferation. In the gut microbiome, carotene supplementation appeared to alter the gut microbiota composition. However, no significant changes were observed in the short-chain fatty acids (SCFA) levels, such as acetic acid, butyric acid, and propionic acid. Furthermore, the differential abundance analysis showed that carotene supplementation reduced the levels of SCFA producers (Odoribacter and Monoglobus genera) in unvaccinated mice compared to the control group, while it enriched the level of SCFA producers (Ruminococcaceae family) and reduced pathobiont levels, commensal bacteria that have pathogenic potential (Mucispirillum genus), in the vaccinated group.