Recent evidence highlights the microbiome’s pivotal role in mucosal immunity via MR1-mediated activation of mucosal-associated invariant T (MAIT) cells, driven by microbial riboflavin biosynthesis intermediates such as 5-OP-RU. While extensively studied in the gut, the relevance of this axis in the female reproductive tract (FRT) remains poorly understood. Yet, MAIT cells have been identified in the FRT, and (microbiome derived) riboflavin is deeply involved in women’s health, as recently reviewed by Dricot et al. (2024). Here, we explore the interplay between vaginal microbiota, riboflavin metabolism, and MAIT cell activation. We have combined targeted metabolomics and 16S rRNA sequencing in a subset of the Isala cohort (n = 257), revealing a significant association between vaginal vitamin B2 levels and dominance by Lactobacillus crispatus, a species associated with optimal vaginal health. These findings were validated using the VIRGO2 metagenomic/metatranscriptomic dataset, confirming in vivo riboflavin production, especially in L. crispatus-dominated vaginal profiles. To assess its functional relevance, we isolated riboflavin-overproducing Lactobacillaceae, such as Limosilactobacillus reuteri strain A (0.356 mg/mL) and B (16.459 mg/mL), and included them in dedicated in vitro experiments. Co-culture with vaginal epithelial cells (VK2, VK2.hMR1) induced MR1 surface expression and altered intracellular riboflavin levels and redox state. Immune profiling using 2D/3D epithelial models, qPCR and Olink proteomics showed enhanced IL-7, IL-12, and IL-18 responses to overproducing strain B compared to regular producer strain A, potentially identifying MAIT cell co-stimulatory signals. Furthermore, activation markers IL18Ra and CD137 were upregulated, while cytotoxic CD71 and CD107b were downregulated on peripheral MAIT cells after co-culture with strain B. Together, these findings unveil a novel mechanism by which vaginal lactobacilli modulate mucosal immunity through riboflavin-mediated MAIT cell activation, with implications for barrier homeostasis and host defense. This work opens new avenues for microbiome-based strategies to support female reproductive health through targeted enhancement of B-vitamins production and epithelial-immune interactions.