Heat shock protein-27 (HSP27) is a member of the small HSP family which has been linked to the nuclear factor-kappa B (NF-κB) signaling pathway regulating inflammatory responses. Clinical reports have suggested that low-level light therapy/laser irradiation (LLLT) could be an effective alternative treatment to relieve inflammation during bacterial infection associated with periodontal disease. However, it remains unclear how light irradiation can modulate the NF-κB signaling pathway. We examined whether or not 635 nm irradiation could lead to a modulation of the NF-kB signaling pathway in HSP27-silenced cells and analyzed the functional cross-talk between these factors in NF-κB activation. The results showed that 635 nm irradiation led to a decrease in the HSP27 phosphorylation, reactive oxygen species (ROS) generation, I-κB kinase (IKK)/inhibitor of κB (IκB)/NF-κB phosphorylation, NF-κB p65 translocation and a subsequent decrease in the COX-1/2 expression and prostaglandin (PGE2) release in lipopolysaccharide(LPS)-induced human gingival fibroblast cells (hGFs). However, in HSP27-silenced hGFs, no obvious changes were observed in ROS generation, IKK/IκB/NF-κB phosphorylation, NF-κB p65 translocation, nor in COX-1/2 expression, or PGE2 release. This could be a mechanism by which 635 nm irradiation modulates LPS-induced NF-κB signaling pathway via HSP27 in inflammation. Thus, HSP27 may play a role in regulating the anti-inflammatory response of LLLT.