Exosomes are nano-vesicles that contribute to the effectiveness of many treatments. The aim of this study was to identify profiles of microRNA (miRNA) contained in serum exosomes that are differentially regulated in patients with prostate cancer undergoing carbon ion radiotherapy (CIRT). RNA was extracted from serum exosomes of eight patients with localized prostate cancer before and after CIRT, and miRNA was analyzed by the next generation sequencing. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the major signaling pathways associated with the proliferation of prostate cancer cells, such as MAPK, PI3K-AKT, mTOR, and AMPK may be implicated in the mechanism of CIRT action. Notably, 57 miRNAs present in serum exosomes were significantly altered after application of CIRT. A high pre-CIRT expression level of specific miRNAs (miR-493-5p, miR-323a-3p, miR-411-5p, miR-494-3p, miR-379-5p, miR-654-3p, miR-409-3p, miR-543, and miR-200c-3p) predicted therapeutic benefit of CIRT (P < 0.05). Post-CIRT expression of miR-654-3p and miR-379-5p was also associated with CIRT efficacy (P < 0.05). These results suggest that the anti-prostate cancer mechanisms elicited by CIRT at the molecular level may involve exosomal miRNAs. Furthermore, specific miRNAs in serum exosomes, particularly miR-654-3p and miR-379-5p, may serve as promising non-invasive biomarkers predicting efficacy of CIRT for prostate cancer.