Acute kidney injury (AKI) during Escherichia coli (E. coli) infection presents significant clinical challenges, with evidence suggesting small extracellular vesicles (sEVs) drived miRNAs play a key role in its pathogenesis. This study investigated the impact of sEVs drived miR-128-3p from myeloid-derived suppressor cells (MDSCs) on AKI onset during E. coli infection. Using a rat model induced by transurethral inoculation with a virulent E. coli strain, AKI severity was assessed by measuring blood urea nitrogen (BUN) and serum creatinine (SCr). Mass cytometry evaluated immune cell composition in renal tissues, and pro-inflammatory markers (E-selectin, ICAM1, IL-12, IL-10, p-p65, IκB) were quantified. SEVs were isolated through differential centrifugation and characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The role of sEVs was further examined by inhibiting their production in vitro with GW4869. High-throughput sequencing identified differentially expressed miRNAs, with qRT-PCR confirming elevated miR-128-3p in AKI samples. Results showed that MDSC accumulation during E. coli infection increased exosome release and upregulated miR-128-3p. This miRNA exacerbated AKI by promoting endothelial inflammation and activating the NF-κB pathway. These findings underscore MDSC-derived sEVs drived miR-128-3p as a mechanistic driver in AKI and a potential diagnostic and therapeutic target for E. coli-induced AKI.