Abstract The objective of the present study was to determine the immunomodulatory role of thymoquinone (TQ) regarding its effect on the production of nitric oxide (NO) by rat peritoneal macrophages. Under certain conditions, macrophages and certain other cells can produce high concentrations of NO from its precursor L-arginine via inducible nitric oxide synthase (iNOS) pathway. TQ has been established as the major component of the oil extracted from Nigella sativa plant seeds, which is being used frequently in herbal medicine. TQ (IC50 1.4–2.76 AM) dose- and time-dependently reduced nitrite production, a parameter for NO synthesis, in supernatants of lipopolysaccharide (LPS)-stimulated (5 Ag/ml) macrophages without affecting the cell viability. The protein level of iNOS in peritoneal macrophages was also decreased by TQ in a concentration-dependent manner. In addition, TQ inhibited the increase in iNOS mRNA expression induced by LPS indicated by reverse transcriptionpolymerase chain reaction (RT-PCR). These inhibitory effects of TQ were confirmed by immunofluorescence staining of iNOS in macrophages which showed decreased immunoreactivity for iNOS after treatment with TQ if compared with the control LPSstimulated cells. These results suggest that TQ suppresses the production of NO by macrophages; an effect which may be useful in ameliorating the inflammatory and autoimmune conditions.