The aim of the present study was to determine whether timing of insulin administration influences the hepatic and serum proinflammatory and anti-inflammatory cytokines during endotoxemia stimulated by lipopolysaccharide (LPS). Eighty-one male Sprague-Dawley rats were divided into different time groups and insulin was given 30 min pre-LPS administration or hour 0, 1, 3, 6, 12, 24 after the induction of endotoxemia, respectively. Hepatic and serum proinflammatory cytokines IL-1β, IL-6, and TNF-α, and anti-inflammatory cytokine IL-10 were detected 24 and 48 h after the induction of endotoxemia. Compared with sham control rats, serum concentrations of proinflammatory cytokines IL-1β, IL-6, and TNF-α and anti-inflammatory cytokine IL-10 significantly increased on 24 and 48 h after induction of endotoxemia. Similarly, LPS administration also significantly increased the hepatic IL-1β, TNF-α, IL-6, and IL-10 protein concentration 48 h after LPS injection. Compared with levels in positive LPS controls animals receiving saline, on 24 and 48 h after LPS injection, insulin administrated ahead of 6 h after LPS injection significantly decreased the serum IL-1β, IL-6, and TNF-a concentration (P < 0.05), and significantly increased anti-inflammatory cytokine IL-10 concentration (P < 0.05); hepatic IL-1β and IL-6 expression were (P < 0.05) significantly decreased compared with levels in positive LPS controls. But, the significant decrease of hepatic TNF-a expression and significant increase of hepatic IL-10 were only seen in the animals in which insulin was administrated at 30 min pre-LPS or coadministrated with LPS. Insulin administrated 6 h after LPS injection lost the ability to significantly reduce serum or hepatic IL-1β, TNF-α, and IL-6 concentrations. Insulin has a protective role in systemic inflammatory response syndrome related to sepsis, such as downregulation of proinflammatory cytokines and upregulation of anti-inflammatory cytokine production. However, timing of insulin administrated may change its effect of inflammatory response in endotoxemic rats. Insulin administrated 6 h after LPS injection weaken the ability to protect inflammatory response related to sepsis.