Milk contains microRNAs (miRNA) that are shielded by small extracellular vesicles (sEVs). Beyond variations among individuals, many factors including nutrition play a role in shaping miRNA expression profiles. This study is to explore milk-derived sEVs-miRNA variations induced by inulin supplementation in subclinical mastitis-suffering cows. Fourteen lactating cows diagnosed with subclinical mastitis were equally assigned to either an inulin or a control group. Apart from total mixed rations, cows in the inulin group were provided with 300 g/d inulin during the morning feeding, while the control group did not receive any supplement. Following 1 wk of adaptation and 5 wk of treatment, sEVs-miRNA were isolated from the milk of each cow. RNA is subjected to high-throughput sequencing and differentially expressed (DE) miRNA (P < 0.05 and ∣ log2FC∣> 1) were detected through bioinformatics analysis. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to examine the target genes of DE miRNA. A sum of 350 miRNA was discovered, including 332 in the control group and 249 in the inulin group. Among these, 9 miRNA showed differential expression within the 2 groups, including 3 upregulated and 6 downregulated in the inulin group. The DE miRNA participates in regulating organismal systems, cellular processes, and signal transduction, which may affect inflammatory response and milk production. Overall, our study provides insight into the micromolecular-level mechanism of inulin in alleviating subclinical mastitis in dairy cows.