MicroRNAs (miRNAs) are widely known as critical regulators in isoflurane-induced neurotoxicity during the development of brain. Moreover, isoflurane could aggravate cognitive impairment in diabetic rats. The present study was designed to investigate the role and mechanism of miR-140-5p on isoflurane-induced neurotoxicity in diabetic rats. Firstly, a diabetic rat model was established by injection of streptozotocin (STZ) and identified by Morris water maze test. The result indicated that isoflurane treatment exacerbated STZ-induced cognitive impairment, as demonstrated by increase of the latency to the platform and decrease of the proportion of time spent in the target quadrant. Secondly, miR-140-5p was up-regulated in diabetic rats treated with isoflurane. Functional assays revealed that knockdown of miR-140-5p attenuated neurotoxicity in diabetic rats, which was shown by a decrease of the latency to the platform and an increase of the proportion of time spent in the target quadrant. Mechanistically, we demonstrated that miR-140-5p directly bonded to SNX12 (sorting nexin 12). At last, the neuroprotective effect of miR-140-5p knockdown against isoflurane-aggravated neurotoxicity in diabetic rats was dependent on up-regulation of SNX12 and inhibition of cell apoptosis. In summary, these meaningful results demonstrated the mitigation of miR-140-5p knockdown against isoflurane-aggravated neurotoxicity in diabetic rats via SNX12, suggesting a novel target for neuroprotection in diabetes under isoflurane treatment.