Extracellular vesicles (EVs) are secreted by various tissues and cells under normal physiological or pathological conditions. Exercise-induced EVs may be involved in the adaptation of exercise-induced fatigue. The 1500-m freestyle is the longest pool-based swimming event in the Olympic Games, and there is a paucity of information regarding changes in the miRNA profiles of circulating EVs after a single session of fatiguing swimming. In this study, 13 male freestyle swimmers conducted a fatiguing 1500-m freestyle swimming session at the speed of their best previously recorded swimming performance. Fasting venous blood was collected before and after the swimming session for analysis. 70 miRNAs from the circulating EVs were found to be differentially expressed after the fatiguing 1500-m freestyle swimming session, among which 45 and 25 miRNAs were up-regulated and down-regulated, respectively. As for the target genes of five miRNAs (miR-144-3p, miR-145-3p, miR-509-5p, miR-891b, and miR-890) with the largest expression-fold variation, their functional enrichment analysis demonstrated that the target genes were involved in the regulation of long-term potentiation (LTP), vascular endothelial growth factor (VEGF), glutathione metabolism pathway, dopaminergic synapse, signal transmission, and other biological processes. In summary, these findings reveal that a single session of fatiguing swimming modifies the miRNAs profiles of the circulating EVs, especially miR-144-3p, miR-145-3p, miR-509-5p, miR-891b, and miR-890, which clarifies new mechanisms for the adaptation to a single session of fatiguing exercise from the perspective of EV-miRNAs.