The phosphatidylethanolamine (PE)-binding proteins (PEBPs) are an evolutionarily conserved family of proteins with pivotal biological functions. Here we describe the cloning and functional characterization of a novel family member, human phosphatidylethanolamine-binding protein 4 (hPEBP4). hPEBP4 is expressed in most human tissues and highly expressed in tumor cells. Its expression in tumor cells is further enhanced upon tumor necrosis factor (TNF) α treatment, whereas hPEBP4 normally co-localizes with lysosomes, TNFα stimulation triggers its transfer to the cell membrane, where it binds to Raf-1 and MEK1. L929 cells overexpressing hPEBP4 are resistant to both TNFα-induced ERK1/2, MEK1, and JNK activation and TNFα-mediated apoptosis. Co-precipitation and in vitro protein binding assay demonstrated that hPEBP4 interacts with Raf-1 and MEK1. A truncated form of hPEBP4, lacking the PE-binding domain, maintains lysosomal co-localization but has no effect on cellular responses to TNFα. Given that MCF-7 breast cancer cells expressed hPEBP4 at a high level, small interfering RNA was used to silence the expression of hPEBP4. We demonstrated that down-regulation of hPEBP4 expression sensitizes MCF-7 breast cancer cells to TNFα-induced apoptosis. HPEBP4 appears to promote cellular resistance to TNF-induced apoptosis by inhibiting activation of the Raf-1/MEK/ERK pathway, JNK, and PE externalization, and the conserved region of PE-binding domain appears to play a vital role in this biological activity of hPEBP4.