Pyridoxal (PL) has been shown to suppress lipopolysaccharide (LPS)-induced gene expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Commonly used cell culture media contain extremely high concentrations of pyridoxine (PN) compared to total serum levels of vitamin B6. Therefore, we evaluated how physiological concentrations of PN influence LPS-stimulated gene expression of COX-2 and iNOS. The mouse macrophage cell line, RAW264.7, was cultured in PN-free DMEM supplemented with 10% fetal bovine serum (DMEM(-PN+FBS)) for 7 d. Although the level of pyridoxal 5'-phosphate in these cells was decreased by 65%, no change was observed in cell proliferation rate or aspartate aminotransferase activity for 7 d. LPS-induced expression of COX-2 mRNA was compared between DMEM(+FBS) and DMEM(-PN+FBS). COX-2 expression was enhanced by 2.2 or 1.9 times with a 1 or 3 d treatment, respectively; however, no difference was observed at 7 d. PN (0.032-100 μm) added to the DMEM(-PN+FBS) and RAW264.7 cells was cultured in the medium containing each concentration of PN for 1 d. Enhancement of COX-2 and iNOS gene expression was suppressed by PN addition in a concentration-dependent manner. COX-2 and iNOS mRNA were similarly expressed in cells grown in media containing PN at 4 μm or higher. Overall, induction of COX-2 and iNOS by LPS was transiently enhanced when RAW264.7 cells were cultured in physiological PN concentrations.