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This study explored the effects over time of lactic acid (LA) on I κBα phosphorylation and nuclear factor-kappa B (NF-κB) p65 protein expression, and on tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNA levels in rat intestinal mucosa microvascular endothelial cells (RIMMVECs) stimulated by lipopolysaccharide (LPS). IκBα, phosphorylated IκBα (ρ-I κBα) and p65 protein levels were monitored by Western blot analysis, and TNF-α and IL-6 mRNA levels were analyzed using real-time PCR. LA treatment reduced TNF-α and IL-6 mRNA levels in LPS-stimulated RIMMVECs, with the greatest effect being after 3 h. The highest inhibitory effect of LA on I κBα phosphorylation to prevent activation of NF- κB was after 6 h. These results suggest that LA reduces TNF-α and IL-6 mRNA levels through decreasing IκBα phosphorylation and blocking the dissociation of IKK complex, which prevents activation of NF- κB.