In recent years, the association between microRNAs (miRNAs) and autophagy in cerebral infarction (CI) has attracted increasingly more attention. The mammalian target of the rapamycin (mTOR) pathway is a key protein regulating the autophagy response. miR-100-5p can bind to the mTOR protein, but its role in CI remains unclear yet. This experiment aims to clarify the role of miR-100-5p in CI. Bioinformatics analysis was performed to screen differentiated expressed functional genes between CI tissue and normal tissue specimens. In vivo experiments: the mouse model of CI was established by middle cerebral artery occlusion (MCAO) methods, After being treated with miR-100-5p-overexpressing lentivirus, the amount of terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL)-positive fluorescence and the fluorescent expression level of mTOR protein were significantly inhibited in the CI region. Western blotting analysis showed that miR-100-5p inhibited the protein expression level of phosphorylated mTOR and total mTOR and enhanced the expression of autophagy-related proteins Beclin, microtubule-associated protein light chain 3II (LC-3II), and autophagy-related gene 7 (ATG-7). For in vitro experiment, after the BV-2 cells were successfully infected with the control lentivirus and miR-100-5p-overexpression lentivirus, they were stimulated with 1% hypoxia and low-glucose medium in a tri-gas incubator for 24 h. It was found that miR-100-5p could significantly lower the protein expression level of phosphorylated mTOR and total mTOR, and increase the expression of the Beclin, LC-3II, ATG-7 autophagy related proteins. miR-100-5p promotes the autophagy response through binding to mTOR protein, thereby inhibiting apoptosis and delaying the progression of CI.