MicroRNA-196a-3p (miR-196a-3p) is known to increase in rat models subjected to middle cerebral artery occlusion (MCAO). In the present work, we aimed to investigate the effects of miR-196a-3p in cerebral ischemia/reperfusion (I/R) injury. Transient cerebral ischemia was induced in C57/BL6 mice subjected to MCAO. Mice were also administered miR-196a-3p antagomir by intracerebroventricular injection. MiR-196a-3p and its target ARF4 levels were quantified and cerebral infarct volume and neuronal apoptosis were evaluated. Primary neural stem cells (NSCs) were activated by oxygen–glucose deprivation/reoxygenation. NSCs were transfected with miR-196a-3p mimics, inhibitors, siARF4, or negative control using Lipofectamine 2000 reagent. ARF4, Ki-67, and Nestin expression levels were assessed using qRT-PCR and western blotting. The proliferation of NSCs was detected by CCK-8 assay and EdU staining. We found that levels of miR-196a-3p expression increased in vivo and in vitro when expression levels of its target ARF4 were decreased. We also found that miR-196a-3p aggravated cerebral I/R injury in vivo. We established that ARF4 is the target of miR-196a-3p using a dual-luciferase assay in vitro. Simultaneously, we observed that miR-196a-3p overexpression or the inhibition of ARF4 inhibited NSC proliferation. MiR-196a-3p inhibited NSC proliferation and aggravated cerebral I/R injury by targeting ARF4.