The enlarged spleen in liver cirrhosis is considered to play a role in the pathogenesis of portal hypertension, but the splenic hemodynamics and molecular mechanisms behind the phenomenon have not been elucidated. The present study aimed to examine the splenic hemodynamics associated with splenic microcirculation and congestion, and to determine the status of the endothelial nitric oxide synthase (eNOS) signaling pathway in the spleen of rats with liver cirrhosis. Liver cirrhosis was induced by bile duct ligation. In rats with bile duct ligation (BDL rats) and control rats, splenic blood flow was measured using a laser Doppler flowmeter, and splenic blood volume was measured using a near-infrared spectrophotometer. The expressions of eNOS and its upstream effectors, Akt, TNF-alpha and VEGF, in the spleen were also determined. Specific splenic blood flow was significantly decreased in BDL rats compared with control rats. Specific splenic blood volume was also decreased in BDL rats, while their total splenic blood volume, especially the deoxygenated volume, was significantly increased. The expressions of phosphorylated and total eNOS, and the eNOS phosphorylation ratio, were all significantly decreased in the spleen of BDL rats. The Akt phosphorylation ratio and TNF-alpha concentration were also decreased in the spleen of BDL rats although the expression of VEGF was increased. These findings suggest that the eNOS signaling pathway is suppressed in the spleen of cirrhotic rats, and may contribute to the measured decreases in specific blood flow and volume in the spleen of liver cirrhosis. Determination of the factors influencing the suppression of eNOS in the spleen may shed light on how liver cirrhosis results in hypodynamic intrasplenic circulation.