4-Hydroxynonenal (HNE) is a pro-apoptotic electrophile generated during the spontaneous decomposition of oxidized lipids. We have previously shown that HNE activates the transcription factor, heat shock factor 1 (HSF1), and promotes cytoprotective heat shock gene expression and that silencing HSF1 sensitizes the colon cancer cell line RKO to HNE-induced apoptosis. Here we report a reduction in the anti-apoptotic proteins Bcl-X(L), Mcl-1, and Bcl-2 in HSF1-silenced RKO cells, and we examine the underlying mechanism. To investigate the regulation of the Bcl-2 family by HSF1, microarray analysis of gene expression was performed. We observed that the Hsp70 co-chaperone, BAG3 (Bcl-2-associated athanogene domain 3), is strongly induced by HNE in control but not in HSF1-silenced colon cancer cells. Silencing BAG3 expression with small interfering RNA caused a dramatic reduction in Bcl-X(L), Mcl-1, and Bcl-2 protein levels in colon cancer cells and increased apoptosis, similar to the effect of silencing HSF1. Also, immunoprecipitation experiments indicate specific interactions between BAG3, Hsp70, and the Bcl-2 family member, Bcl-X(L). Overall, our data reveal that BAG3 is HSF1-inducible and has a unique role facilitating cancer cell survival during pro-apoptotic stress by stabilizing the level of Bcl-2 family proteins.