Background <p>The function of long non-coding RNAs (lncRNAs) in osteoarthritis is increasingly recognized. CKMT2-AS1 has been reported to be dysregulated in osteoarthritis, but its biological function remains unknown.</p> Objective <p>The objective is to investigate the expression and function of CKMT2-AS1 in osteoarthritis, providing a biomarker and a promising therapeutic target.</p> Methods <p>Expression of CKMT2-AS1 was determined in human osteoarthritic cartilage and IL-1β-induced chondrocyte models. Gain-of-function experiments were conducted by overexpressing CKMT2-AS1 in CHON-001 cells. Inflammatory cytokines (IL-6, TNF-α) and extracellular matrix (ECM)-related markers (ACAN, COL2A1, MMP3, MMP13) were assessed via ELISA and RT-qPCR, respectively. EdU, CCK-8, and flow cytometry assays were employed to evaluate cell proliferation and apoptosis. The interaction between CKMT2-AS1 and miR-6720-5p was validated by luciferase reporter assay and RIP assay. Bioinformatics tools predicted the targets of miR-6720-5p, which underwent GO and KEGG enrichment analysis.</p> Results <p>CKMT2-AS1 was downregulated in osteoarthritic cartilage and IL-1β-induced chondrocytes. Overexpression of CKMT2-AS1 alleviated IL-1β-induced inflammation, ECM degradation, proliferation inhibition, and apoptosis. Mechanistically, CKMT2-AS1 acted as a molecular sponge for miR-6720-5p, which was upregulated in osteoarthritis. The functional role of the CKMT2-AS1/miR-6720-5p axis was validated through rescue experiments. miR-6720-5p overexpression effectively reversed the protective effect of CKMT2-AS1. Additionally, the target genes of miR-6720-5p were enriched in pathways such as “Hippo signaling” and “arginine biosynthesis.”</p> Conclusions <p>This study reveals the downregulation of CKMT2-AS1 in osteoarthritis and its protective role against chondrocyte injury via miR-6720-5p, highlighting its novel function as a potential therapeutic target for osteoarthritis.</p>