Background/Objectives: Emerging evidence shows long non-coding RNAs (lncRNAs) as critical regulators of osteoarthritis (OA) progression, often acting in complex networks with microRNAs (miRNAs). In our study, we investigated the potential regulatory function of the lncRNA MEG3/miR-21-5p axis in the OA phenotype of chondrocytes. Methods: Differential gene expression analysis in damaged vs. intact cartilage was performed, re-analyzing existing public RNA-seq data. MiRTarBase, LncRNADisease, and Open Targets databases were utilized to identify miR-21-5p target genes and OA-associated lncRNAs and genes. Functional enrichment analysis and protein–protein interaction (PPI) network construction were performed using the DAVID and STRING databases, respectively. MEG3, miR-21-5p, SOX5, COL2A1 and ACAN mRNA expressions were assessed by qRT-PCR. The role of the MEG3/miR-21-5p axis in OA chondrocytes was examined using transfection experiments. Results: Eighty-one lncRNAs displayed significant differences in expression between damaged and intact cartilage, including MEG3. Bioinformatic analysis indicated that MEG3 interacts with miR-21-5p, while SOX5 was identified to be a putative target of miR-21-5p. MEG3 and SOX5 expression levels were significantly downregulated in OA chondrocytes, whereas miR-21-5p expression was upregulated. Silencing of MEG3 resulted in increased miR-21-5p levels in chondrocytes. Conversely, inhibition of miR-21-5p led to increased SOX5 expression and anabolic markers COL2A1 and ACAN. Notably, MEG3 silencing significantly reduced SOX5 expression, an effect that was reversed upon miR-21-5p inhibition. Conclusions: Our findings highlight a potential regulatory role of the dysregulated MEG3/miR-21-5p axis in modulating the anabolic phenotype of chondrocytes through regulation of SOX5 expression. This novel lncRNA/miRNA/mRNA regulatory network may represent a candidate therapeutic axis for knee osteoarthritis.