Gut microbes play a vital role in maintaining homeostasis through a continuous interaction with our mucosal immune system, yet the key commensal microbes that restore or protect health from disease remain largely unclear. This lack of clarity limits the development of effective microbial therapies, including probiotic treatments, which have been made more challenging due to an incomplete understanding of the molecular mechanisms underlying their immunomodulatory properties. This project addresses this gap of knowledge and explores the stages by which the probiotic bacterium Lactiplantibacillus plantarum (LP) induces innate immune responses, focusing on the elevated production of type I interferon (IFN-I) cytokines in macrophages. IFN-I cytokines are essential to combat microbial infections and modulate autoimmune responses. Such enhanced responses were found to be strain-specific and dependent on the ability of LP to express adhesins and self-aggregate. Interestingly, the self-aggregation phenotype was found to be regulated by the monosaccharides metabolised by LP and this was particularly essential for an enhanced interaction with macrophages, thus influencing the number of bacteria phagocytosed. Furthermore, through the use of receptor specific blocking antibodies and inhibitors, the C-type lectin receptor Mincle was observed to be involved in LP uptake and its subsequent intracellular processing, resulting in the activation of IFN-I associated responses that were mainly mediated by the cytosolic DNA sensor cGAS. In this respect, sensing of LP genomic DNA was also regulated by the sugar metabolism of LP due to its influence on DNA methylation, which was an additional factor for prominent IFN-I responses. Overall, the findings demonstrate that self-aggregation and DNA methylation are two key immunomodulatory features of LP, both intrinsically linked to its nutritional (sugar) preferences. Thus, elucidating these interconnected mechanisms could pave the way for the development of targeted probiotic formulations with specific strains and sugars, thereby manipulating host-microbe interactions and offering novel immunomodulatory approaches against disorders affecting the gut mucosa.