Abstract Body (Do not enter title and authors here): Background: Acute ischemic stroke (AIS) is one of the leading causes of death and disability in adults worldwide, and effective treatments are still lacking. Cold-inducible RNA-binding protein (CIRP) is up-regulated in response to various stress conditions. Previous studies have indicated that CIBP could potentially influence the development of various diseases. Research questions: Nevertheless, the specific role and molecular mechanism of CIBP after AIS remain unclear. Methods: The levels of serum CIRP and other brain injury markers such as NSE, S100β, GSDMD, and NLRP3 were measured by ELISA to explore the relationship between CIRP and AIS. Additionally, middle cerebral artery occlusion models (MCAO) in rats were established to investigate its localization in brain tissue using immunofluorescence staining. Additionally, the CIRP knockout MCAO rat model was established, and evaluate the effect of CIRP deficiency on the neurological function of rats. while the role of CIRP was investigated through RNA sequencing and Western Blot analysis. Additionally, the potential protective effect of the CIRP inhibitor C23 on neural injury following AIS was examined. Results: (1) Compared to the healthy control group, the serum levels of CIRP and other brain injury markers (NSE, S100β) in AIS patients and MCAO rats were significantly elevated, and the elevated levels were positively correlated. (2) In the MCAO rat model, the CIRP was mainly located in neurons and microglia. (3) Compared with wild-type MCAO rats, CIRP knockout MCAO rats improved neurological function scores. (4) RNA-seq analysis revealed that genes differentially expressed between wild-type MCAO rats and CIRP knockout MCAO rats were enriched in inflammation-related pathways. (5) The expression of pyroptosis-related proteins (NLRP3, Caspase-1, GSDMD, and ASC) and inflammatory factors (IL-1β, TNF-α, etc.) decreased in CIRP knockout MCAO rats. (6) The CIRP inhibitor C23 significantly alleviated brain injury and improved neurological function. C23 treatment significantly decreased the expression levels of pyroptosis-related proteins and inflammatory factors. Conclusions: This study shows that CIRP plays an important role in AIS development. The levels of CIRP in AIS patients and MCAO rats are significantly increased and are positively correlated with nerve damage markers. Thus, supporting CIRP as a potential target for the treatment of AIS. This discovery provides new strategies for the treatment of AIS.