Alpha-lipoic acid (ALA) is a known anti-inflammatory agent that can be used as a pharmacological agent in adjunctive therapy to inhibit the recurrence of periodontitis. This study investigated the inflammatory regulation effect of pre-administered ALA on human gingival fibroblasts (HGFs) stimulated with Escherichia coli-derived lipopolysaccharide (LPS). HGFs were administered ALA and then stimulated with LPS. Western blot analysis was used to investigate activation of the nuclear factor-kappa B (NF-κB) signaling pathway. Analysis of NF-κB p65 nuclear translocation was performed using an immunofluorescence, assay and inflammatory cytokine (TNF-α, IL-1β, IL-6, and IL-8) secretion analysis was conducted using an enzyme-linked immunosorbent assay. NF-κB signaling pathway expression was increased by LPS stimulation. However, increased NF-κB signaling pathway expression was down-regulated by ALA pre-administration. Further, NF-κB p65 was translocated to the nucleus by LPS stimulation. However, NF-κB p65 translocation was inhibited by ALA pre-administration. The secretion of TNF-α, IL-1β, IL-6, and IL-8 was increased by LPS stimulation but down-regulated by ALA pre-administration. The results of this study demonstrated that ALA regulated the secretion of inflammatory cytokines via regulation of NF-κB signaling pathway activation in HGFs, which suggests that ALA has the potential to regulate periodontal tissue inflammation.