Vasoplegia impedes therapeutic interventions to restore vascular tone, leading to severe hypotension, poor tissue perfusion, and multiple organ failure in septic shock. High levels of circulating nitric oxide (NO) play a crucial role in endotoxin-induced vascular hyporeactivity. Proinflammatory cytokines have been implicated in the induction of inducible NO synthase and overproduction of NO. Anti-inflammatory therapy can diminish NO formation and improve vascular hyporeactivity in septic shock. STE20/SPS1-realted proline/alanine-rich kinase (SPAK) has been reported to activate mitogen-activated protein kinase and contribute to intestinal inflammation. Thus, we evaluated the roles of SPAK in NO production and vascular hyporeactivity in endotoxemic animals. Male wild-type and SPAK deficiency mice were intraperitoneally administered vehicle or Escherichia coli lipopolysaccharide (LPS, 50 mg/kg). The changes of systolic blood pressure and plasma nitrate and nitrite levels were measured during the experimental period. Thoracic aortas were exercised to assess vascular reactivity and SPAK expression. In the present study, mice in endotoxin model showed severe hypotension and hyporeactivity to serotonin, phenylephrine (PE), and acetylcholine in the aortic rings. Phosphorylated SPAK expression in the aorta and NO levels in the plasma were also increased in animals with endotoxic shock. However, deletion of SPAK not only reduced the elevation of NO levels but also improved vascular hyporeactivity to serotonin and PE in endotoxemic mice. Taken together, SPAK could be involved in the NO overproduction and vascular hyporesponsiveness to vasoconstrictors in endotoxic shock. Thus, inhibition of SPAK could be useful in the prevention of endotoxin-induced vascular hyporeactivity.