Lipid peroxidation is a consequence of both normal physiology and oxidative stress that generates various reactive metabolites, a principal end product being 4-hydroxynonenal (HNE). As a diffusible electrophile, HNE reacts extensively with cellular nucleophiles. Consequently, HNE alters cellular signaling and activates the intrinsic apoptotic cascade. We have previously demonstrated that in addition to promoting apoptosis, HNE activates stress response pathways, including the antioxidant, endoplasmic reticulum stress, DNA damage, and heat shock responses. Here we demonstrate that activation of the heat shock response by HNE is dependent on the expression and nuclear translocation of heat shock factor 1 (HSF1), which promotes the expression of heat shock protein 40 (Hsp40) and Hsp70-1. Ectopic expression and immunoprecipitation of c-Myc-tagged Hsp70-1 indicates that HNE disrupts the inhibitory interaction between Hsp70-1 and HSF1, leading to the activation heat shock gene expression. Using siRNA to silence HSF1 expression, we observe that HSF1 is necessary for the induction of Hsp40 and Hsp70-1 by HNE, and the lack of Hsp expression is correlated with an increase in apoptosis. Nrf2, the transcription factor that mediates the antioxidant response, was also silenced using siRNA. Silencing Nrf2 also enhanced the cytotoxicity of HNE, but not as effectively as HSF1. Silencing HSF1 expression facilitates the activation of JNK pro-apoptotic signaling and selectively decreases expression of the anti-apoptotic Bcl-2 family member Bcl-X(L). Overexpression of Bcl-X(L) attenuates HNE-mediated apoptosis in HSF1-silenced cells. Overall, activation of HSF1 and stabilization of Bcl-X(L) mediate a protective response that may contribute significantly to the cellular biology of lipid peroxidation.