Although probiotics have been reported to reduce the gastric inflammatory response to Helicobacter pylori infection, little information is available regarding the molecular mechanisms behind this reduction. This study investigates the role of conjugated linoleic acids (CLA) produced by probiotics in interactions of IkappaB kinase (IKK) and heat shock protein 90 (Hsp90) to activate the nuclear factor-kappaB (NF-kappaB) signaling pathway in human gastric epithelial cells infected with H. pylori. Conditioned medium (CM) containing Lactobacillus acidophilus-producing CLA significantly inhibited the activated NF-kappaB signals and the upregulated expression of interleukin-8 (IL-8) in MKN-45 cells infected with H. pylori. Pretreatment with CM with CLA attenuated the increased IKK activity induced by H. pylori. Transfection of siRNA for IKK-beta dramatically reduced H. pylori-induced IkappaBalpha phosphorylation, but siRNA for IKK-alpha had little effect on IkappaBalpha phosphorylation, although the siRNA for IKK-alpha significantly decreased IL-8 production. Furthermore, Hsp90 was associated with IKK-alpha and IKK-gamma in H. pylori-infected cells, and CM with CLA dissociated the complex between Hsp90 and IKK-gamma. These results suggest that CLA produced by probiotics has anti-inflammatory activity in gastric epithelial cells infected with H. pylori via dissociation of the IKK-gamma and Hsp90 complex.