Spinal cord injury (SCI) represents a relatively common type of motor system trauma. While the SCI patient will experience varying degrees of paraplegia and quadriplegia, which severely affects their quality of life, a heavy burden is also placed on the family and society as a whole. The exact pathogenic mechanisms underlying this condition remain unknown and no specific treatments are currently available. Findings from recent studies have shown that mesenchymal stem cells (MSCs), derived from extracellular vesicles (EVs) can reduce apoptosis, inflammation and promote angiogenesis after SCI. However, the mechanisms through which EVs exert these effects have yet to be identified, indicating the necessity for further investigation. In the present study, we report that treatment with MSCs-EVs significantly improved functional recovery and attenuated lesion size and apoptosis in a rat model of SCI. These MSCs-EVs were found to be directed to the spinal injury site and mainly incorporated into neurons within the lesioned site of the spinal cord. Tandem Mass Tags quantitative proteomics was applied to compare protein changes after SCI and MSCs-EVs treatment. A total of 883 differential proteins were identified, many of which being associated with apoptosis and inflammation. Subsequently, miRNA contents of MSCs-EVs were determined using qRT-PCR, with the result that miR-21-5p was one of the most highly expressed miRNA in these MSCs-EVs. Moreover, inhibition of miR-21-5p in MSCs-EVs significantly reversed the beneficial effects of MSCs-EVs on motor function and apoptosis, an effect which was associated with modulating FasL expression. The data suggest that modulation of the MSCs-EVs miR-21-5p/FasL gene axis may serve as a promising strategy for clinical treatment of SCI and other neurological diseases.