Human periodontal ligament stem cells (hPDLSCs) play a critical role in the regeneration of periodontal tissue. Forkhead box protein A1 (FOXA1) has been implicated in the inflammatory mechanisms of various diseases. However, the role of FOXA1 in periodontal inflammation and its effect on the osteogenic differentiation of hPDLSCs remains unclear. In this study, healthy tooth root-derived hPDLSCs were isolated, and flow cytometry was used to detect cell surface markers. Western blot and immunofluorescence analyses were performed to assess FOXA1 levels in different tissues. The levels of inflammatory factors were measured using Western blot and ELISA kits. Alkaline phosphatase (ALP) staining, alizarin red S staining, and Western blot were employed to evaluate the impact of FOXA1 silencing on the osteogenic differentiation of hPDLSCs. Finally, the protein levels in the Toll-like receptor 4 (TLR4)/Myeloid differentiation factor-88 (MyD88)/NF-κB pathway were analyzed using Western blot. Results showed that periodontal membrane tissues from patients with periodontitis exhibited a marked increase in FOXA1 levels. Lipopolysaccharide (LPS) treatment significantly upregulated FOXA1 expression in hPDLSCs, elevated inflammatory factor levels, and inhibited osteogenic differentiation. However, silencing FOXA1 mitigated the effects of LPS. Furthermore, LPS treatment activated the TLR4/MyD88/NF-κB pathway, while FOXA1 silencing impeded this activation. Notably, the application of the TLR4 agonist CRX-527 reversed the inhibitory effects of FOXA1 silencing on LPS-induced responses. In summary, silencing FOXA1 reduced cellular inflammation by inhibiting the TLR4/MyD88/NF-κB pathway and alleviated the suppressive effects of LPS on the osteogenic differentiation of hPDLSCs.