BACKGROUND: Several long non-coding RNAs (lncRNAs) are dysregulated in chronic periodontitis (CP). PURPOSE: The study aimed to elucidate the molecular mechanisms and clinical significance of lncRNA EPB41L4A antisense RNA 1 (EPB41L4A-AS1) in CP. PATIENTS AND METHODS: This study enrolled 101 patients with CP and 90 subjects with healthy periodontal tissues. Patients with CP were categorized according to severity. The expression of EPB41L4A-AS1 and osteogenic markers in the lipopolysaccharide (LPS)-induced human periodontal ligament cells (hPDLCs) was assessed using real-time quantitative reverse transcription PCR (RT-qPCR). The diagnostic significance of EPB41L4A-AS1 was evaluated using receiver operating characteristic (ROC) analysis. The levels of inflammatory factors were measured using an enzyme-linked immunosorbent assay. Cell proliferation and apoptosis were analyzed using cell counting kit -8 and flow cytometry, respectively. The interaction between EPB41L4A-AS1 and microRNAs was verified using dual luciferase reporter assays, RNA immunoprecipitation, and RNA pull-down assays. RESULTS: EPB41L4A-AS1 was downregulated in the gingival sulcus fluid of patients with CP and LPS-induced hPDLCs. Additionally, EPB41L4A-AS1 could distinguish patients with CP from control subjects with sensitivity (88.12%) and specificity (81.11%). The expression of EPB41L4A-AS1 was downregulated in patients with severe CP. EPB41L4A-AS1 downregulation was directly correlated with severe clinical indicators and inversely correlated with inflammatory indicators. The overexpression of EPB41L4A-AS1 promoted the proliferation and osteogenic differentiation of hPDLCs and mitigated LPS-induced inflammation. Mechanistically, EPB41L4A-AS1 directly targets and downregulates miR-214-3p expression, resulting in the upregulation of Yes1-associated transcriptional regulator (YAP1) levels. The overexpression of miR-214-3p partially suppressed the effects of EPB41L4A-AS1 on LPS-induced hPDLC injury and osteogenic differentiation. CONCLUSION: The overexpression of EPB41L4A-AS1 suppressed LPS-induced hPDLC injury and enhanced osteogenic differentiation through the miR-214-3p/YAP1 axis. Thus, EPB41L4A-AS1 is a novel diagnostic marker and a therapeutic target for CP.