Interleukin‐1β (IL‐1β) is closely involved in liver disorders. IL‐1β produces nitric oxide (NO) in vascular smooth muscle cells and relaxes vascular smooth muscle via cyclic guanosine 3′,5′‐monophosphate (cGMP). In this study, we evaluated the relaxing effect of IL‐1β on cultured Ito cells. Ito cells were isolated from the livers of male Wistar rats and cultured for 24 hours. Immunolocalization of inducible nitric oxide synthase (iNOS) and cGMP and intensity of fluorescence of cGMP were examined using a confocal laser microscope. Ito cells were treated with 0, 200, and 1,000 pmol/L IL‐1β, and the intracellular cGMP concentration was measured after 12 hours. Moreover, Ito cells treated with 200 and 1,000 pmol/L IL‐1β and not treated with IL‐1β were observed over 12 hours, and the area of the same Ito cell was compared before and after the addition of IL‐1β. Next, effects of N^G‐monomethyl‐L‐arginine (L‐NMMA) and S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) on Ito cell relaxation by IL‐1β treatment were examined. In Ito cells, immunofluorescence of iNOS was observed, and fluorescent intensity of cGMP increased after addition of IL‐1β. Intracellular cGMP concentration increased dose‐dependently after addition of IL‐1β. Cell area significantly increased in the IL‐1β‐treated group compared with the untreated group. Relaxation of Ito cells by IL‐1β treatment was inhibited by L‐NMMA in a dose‐dependent manner, but was enhanced by SNAP. These results indicate that IL‐1β produces NO in cultured Ito cells and relaxes the cells via cGMP.