Display omitted •Overexpression of microRNA-9a-5p ameliorates ischemic brain injury.•MicroRNA-9a-5p regulates the expression of NLRP1.•MicroRNA-9a-5p influences the NLRP1 inflammasome activation. The nucleotide oligomerization domain (NOD)-like receptor (NLR) pyrin domain-containing protein 1 (NLRP1) inflammasome has been shown to contribute to brain injury after ischemic stroke. Our previous study showed that microRNA-9a-5p (miR-9a-5p) ameliorates ischemic injury by regulating neuronal autophagy in rats subjected to middle cerebral artery occlusion (MCAO) surgery. The aims of this study were to investigate whether miR-9a-5p can influence the NLRP1 inflammasome following ischemic stroke and to clarify the mechanism involved. We found that MCAO in rats increased the level of NLRP1 inflammasome proteins, including NLRP1 receptor, ASC and precursor caspase-1, which induced higher levels of cleaved caspase-1, mature interleukin-1β (IL-1β) and interleukin-18 (IL-18). Similarly, the levels of the NLRP1 inflammasome proteins, cleaved caspase-1, mature IL-1β and IL-18 were elevated in SY-5Y cells exposed to oxygen-glucose deprivation (OGD). Further investigation showed that NLRP1 was a target of miR-9a-5p and was downregulated by miR-9a-5p overexpression and upregulated by miR-9a-5p inhibition. Moreover, overexpression of miR-9a-5p not only decreased the levels of NLRP1, ASC and precursor caspase-1 but also reduced the levels of IL-1β and IL-18 in MCAO rats and OGD cells. Therefore, we conclude that miR-9a-5p is involved in NLRP1 inflammasome-mediated ischemic injury, which further suggests that the overexpression of miR-9 may be an effective way to ameliorate brain injury following ischemic stroke.