Autoreactive encephalitogenic T cells exist in the healthy immune repertoire but need a trigger to induce CNS inflammation. The underlying mechanisms remain elusive, whereby microbiota were shown to be involved in the manifestation of CNS autoimmunity. Here, we used intravital imaging to explore how microbiota affect the T cells as trigger of CNS inflammation. Encephalitogenic CD4 ⁺ T cells transduced with the calcium-sensing protein Twitch-2B showed calcium signaling with higher frequency than polyclonal T cells in the small intestinal lamina propria (LP) but not in Peyer’s patches. Interestingly, nonencephalitogenic T cells specific for OVA and LCMV also showed calcium signaling in the LP, indicating a general stimulating effect of microbiota. The observed calcium signaling was microbiota and MHC class II dependent as it was significantly reduced in germfree animals and after administration of anti-MHC class II antibody, respectively. As a consequence of T cell stimulation in the small intestine, the encephalitogenic T cells start expressing Th17-axis genes. Finally, we show the migration of CD4 ⁺ T cells from the small intestine into the CNS. In summary, our direct in vivo visualization revealed that microbiota induced T cell activation in the LP, which directed T cells to adopt a Th17-like phenotype as a trigger of CNS inflammation.