Recently, photobiomodulation (PBM) has emerged as a novel therapeutic approach for modulating the gut microbiome, offering potential for the regulation of intestinal inflammation. Although PBM has been primarily used for various clinical applications, recent studies suggest that its effects may extend to the regulation of microbial imbalances and chronic inflammation. The biological effects of PBM are wavelength-dependent, as the wavelength of light determines tissue penetration depth and cellular response. The current study aimed to compare the inflammation-modulatory effects of PBM at four different wavelengths (405, 532, 635, and 808 nm) and to elucidate the underlying molecular mechanisms of PBM in intestinal inflammation. An in vitro co-culture model consisting of Caco-2 cells and Lactobacillus was established to simulate the intestinal environment. Cellular inflammation was induced by lipopolysaccharide (LPS) stimulation, followed by wavelength-dependent PBM treatment at a dosage of 10 J/cm(2) (100 mW/cm(2) for 100 s, applied as a single irradiation). Among the wavelengths, 635 nm significantly reduced nitric oxide production and suppressed the expression of pro-inflammatory cytokines (TNF-alpha, IL-6, IL-1beta, and iNOS). Furthermore, western blot and qPCR analyses revealed that 635 nm PBM downregulated key signaling factors in the MAPK/NF-kB pathway, indicating a potential molecular mechanism for its anti-inflammatory effect. These findings suggest that PBM, particularly at 635 nm, may serve as an effective strategy for modulating intestinal inflammation. Further studies will investigate the anti-inflammation and microbiome modulation effects of PBM in an in vivo model of inflammatory bowel disease.