Background and Aims Carotid plaque instability is a critical pathological basis for ischemic stroke. Identifying key molecular markers to evaluate plaque stability has important clinical implications. Recent studies have emphasized the regulatory roles and predictive value of long non-coding RNAs (lncRNAs) in plaque stability. In our previous transcriptome sequencing analysis of human stable and unstable carotid plaques, we identified lncRNA C-C motif chemokine ligand 3 antisense RNA 1(CCL3-AS1) as significantly upregulated in unstable plaques, suggesting a potential association with plaque instability. Therefore, this study aimed to validate CCL3-AS1 expression in an expanded plaque sample cohort and to explore its role and underlying molecular mechanism in carotid plaque destabilization.Methods Carotid plaque specimens were obtained from patients undergoing carotid endarterectomy and classified into stable and unstable groups (n=15 per group) based on HE and Sirius red staining. qRT-PCR was used to validate the expression of candidate lncRNA CCL3-AS1. The localization and co-expression of CCL3-AS1 with macrophages in plaques were determined by RNA fluorescence in situ hybridization (FISH) combined with immunofluorescence staining. In vitro, THP1-derived macrophages were transduced with lentivirus or treated with antisense oligonucleotides (ASO) to overexpress or knock down CCL3-AS1, respectively, and the expression levels of inflammatory cytokines and matrix metalloproteinases (MMPs) were assessed. In vivo, an unstable carotid plaque model was established by tandem ligation of the right carotid artery in apolipoprotein E-deficient (ApoE^-/-) mice, followed by local overexpression of CCL3-AS1. The effects on plaque morphology, macrophage infiltration, and MMP-9 expression were evaluated. Additionally, bioinformatic prediction using the catRAPID v2.1 omics platform was performed to identify potential RNA-binding proteins interacting with CCL3-AS1. RNA stability assays and RNA-binding protein immunoprecipitation (RIP) were conducted to verify the regulatory mechanism of MMP-9 expression.Results CCL3-AS1 was significantly upregulated in unstable carotid plaques and was predominantly localized to the cytoplasm of plaque-infiltrating macrophages. In vitro, overexpression of CCL3-AS1 markedly increased the expression of MCP-1, TNF-α, IL-1β, iNOS, and MMP-9 in macrophages, whereas knockdown had the opposite effect. In the ApoE^-/- mouse model of unstable carotid plaques, CCL3-AS1 overexpression led to fibrous cap rupture, increased infiltration of pro-inflammatory macrophages, enhanced MMP-9 secretion, and promoted plaque instability. Co-expression analysis revealed a strong correlation between CCL3-AS1 and MMP-9 expression (r=0.89, P=0.001). RNA stability assays demonstrated that CCL3-AS1 delayed the degradation of MMP-9 mRNA. Bioinformatic prediction identified heterogeneous nuclear ribonucleoprotein K (hnRNP-K) as a potential binding partner of CCL3-AS1. RIP and FISH co-localization confirmed the interaction, suggesting that CCL3-AS1 enhances MMP-9 mRNA stability through binding to hnRNP-K, thereby promoting its expression.Conclusion As a macrophage-enriched inflammatory lncRNA, CCL3-AS1 may promote carotid plaque instability by enhancing MMP-9 expression via hnRNP-K-mediated mRNA stabilization. This lncRNA represents a potential molecular target for early intervention and stratification of ischemic stroke.