Aim To investigate the role and mechanism of KLHL21 gene in myocardial infarction (MI) of mice. Methods KLHL21 gene knockout (KO) mice were generated using CRISPR/Cas9 technology, and C57BL/6 wild-type mice were used as controls. Sixty KLHL21 KO mice and 60 wild-type mice were randomly divided into four groups:WT+Sham group (n＝30), WT+MI group (n＝30), KO+Sham group (n＝30) and KO+MI group (n＝30). Postoperative ischemic and infarct areas were assessed using TTC and Evans Blue staining, myocardial injury markers were measured by ELISA, cardiac function was evaluated by ultrasound, and histological changes were examined using HE and Masson staining. Western blot was used to detect proteins related to the nuclear factor-κB (NF-κB) signaling pathway. ResultsKLHL21 protein expression in the myocardial tissue of KO mice was significantly lower than that in WT mice. The infarct area in KO+MI mice was significantly larger than that in WT+MI group. KO+MI mice showed reduced cardiac function compared with WT+MI mice. HE staining revealed myocardial cell loss, liquefactive necrosis, nuclear fragmentation, and significant neutrophil infiltration, while Masson staining showed aggravated fibrosis in KO+MI group. Serum tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), creatine kinase-MB (CK-MB), and cardiac troponin I (cTnI) levels were significantly increased in KO+MI mice compared with WT+MI mice. Western blot analysis showed increased levels of phosphorylated inhibitor of nuclear factor-κB alpha (p-IKBα), P65, and P50, and decreased nuclear factor-κB alpha (IKBα) in KO+MI mice. Conclusion KLHL21 gene plays a preventive role in myocardial infarction in mice, possibly through inhibition of NF-κB signaling pathway activation.