Abstract: Cyclic dimeric adenosine 3′,5′-monophosphate (c-di-AMP) is a newly-discovered second messenger in bacteria and archaea. By directly binding to or affecting the expression of target proteins, c-di-AMP regulates the physiological functions of bacteria, including maintaining osmotic pressure, balancing central metabolism, monitoring DNA damage, and controlling biofilm and spore formation. As a new pathogen-associated molecular pattern (PAMP), it binds to the host pattern recognition receptor (PRR), induces cyclic GMP-AMP synthase (cGAS)-STING signal axis to produce type Ⅰ interferon by activating the stimulator of interferon genes (STING), and promotes the secretion of inflammatory factors through nuclear factor κB (NF-κB) signaling pathway, thereby playing an important role in host immunity to bacterial infection and tumorigenesis. Due to its immunogenicity, c-di-AMP could be used as an immune adjuvant to provide new targets for the development of vaccines. However, the specific mechanism of action of c-di-AMP in host immunity awaits further exploration. Herein, we presented the structure and biological characteristics of c-di-AMP, and summarized the possible mechanism of c-di-AMP’s regulation of host immune response. In addition, we also reported the latest findings on using c-di-AMP as an immune adjuvant in clinical treatment. Research on the function of c-di-AMP and its mechanism of action on host immune response provides new ideas for finding clinical solutions to bacterial resistance, infection control, tumor prevention, and vaccine development in the future.