<p>Carbon dots (CDs) are unintentionally formed during thermal processing of food and are emerging environmental pollutants that may pose health risks. We investigated the reproductive toxicity of food-derived CDs via the gut-testicular axis by exposing male mice to environmentally relevant doses (25 and 100 mg kg⁻¹ d⁻¹) for 15 weeks. Multi-omics analysis (including metagenomics, transcriptomics, and metabolomics) revealed that CDs significantly altered the gut microbiota composition, reducing beneficial bacteria (<i>Akkermansia muciniphila</i>, <i>P</i>&lt;0.01) while increasing pathogenic bacteria (Desulfovibrionaceae, <i>P</i>&lt;0.001). Functional analysis revealed upregulation of the lipopolysaccharide (LPS) biosynthesis pathway (<i>P</i>&lt;0.001) and reduced levels of barrier-protective tryptophan metabolites. Time-series studies established a mechanistic sequence: microbiota disruption (days 1–3), intestinal barrier dysfunction (days 3–5), blood-testis barrier damage (days 5–7), testicular inflammation, and reproductive dysfunction. Dose-dependent testicular toxicity included reduced testosterone synthesis (<i>P</i>&lt;0.001), impaired spermatogonial stem cell maintenance due to downregulation of PLZF, and impaired fertility. Testicular transcriptomics analysis revealed activation of the IL-17 signaling pathway and inhibition of steroidogenesis. This study provides comprehensive evidence that CD induces male reproductive toxicity through microbiota-dependent mechanisms, emphasizing the environmental health implications of dietary nanoparticle exposure.</p>