Oxidative stress is pathogenic in a variety of diseases, but the mechanism by which cellular signaling is affected by oxidative species has yet to be fully characterized. Lipid peroxidation, a secondary process that occurs during instances of free radical production, may play an important role in modulating cellular signaling under conditions of oxidative stress. 4-Hydroxy-trans-2-nonenal (HNE) is an electrophilic aldehyde produced during lipid peroxidation that forms covalent adducts on proteins, altering their activity and function. One such target, LKB1, has been reported to be inhibited by HNE adduction. We tested the hypothesis that HNE inhibits LKB1 activity through adduct formation on a specific reactive residue of the protein. To elucidate the mechanism of the inhibitory effect, HEK293T cells expressing LKB1 were treated with HNE (10 μm for 1 h) and assayed for HNE-LKB1 adduct formation and changes in LKB1 kinase activity. HNE treatment resulted in the formation of HNE-LKB1 adducts and decreased LKB1 kinase activity by 31 ± 9% (S.E.) but had no effect on the association of LKB1 with its adaptor proteins sterile-20-related adaptor and mouse protein 25. Mutation of LKB1 lysine residue 97 reduced HNE adduct formation and attenuated the effect of HNE on LKB1 activity. Taken together, our results suggest that adduction of LKB1 Lys-97 mediates the inhibitory effect of HNE.