Tumor necrosis factor (TNF) is an important mediator that triggers onset of autoimmune diseases and exerts its biological effects by interacting through two receptors, TNFR1 (also known as TNFRSF1A) and TNFR2 (also known as TNFRSF1B). TNFR2 signaling has significant potential to exert pro-survival and protective roles in several diseases. Unlike TNFR1 signaling, however, the mechanism of TNFR2 signal transduction is poorly understood, and few of its adaptor molecules are known. The present study utilized a proteomics approach to search for adaptor molecules in the TNFR2 signaling complex and identified aminopeptidase P3 (APP3, also known as XPNPEP3) to be a key molecule. One of its two isoforms, mitochondrial APP3 (APP3m) but not cytosolic APP3 (APP3c), was recruited to TNFR2 and shown to regulate TNF-TNFR2-dependent phosphorylation of JNK1 (also known as MAPK8) and JNK2 (also known as MAPK9). Furthermore, APP3m was released from mitochondria upon TNF stimulation in the absence of mitochondrial outer membrane permeabilization (MOMP). The observation of increased cell death upon downregulation of APP3m also suggested that APP3m exerts an anti-apoptotic function. These findings reveal that APP3m is a new member of the TNF-TNFR2 signaling complex and characterize an APP3-mediated TNFR2 signal transduction mechanism that induces activation of JNK1 and JNK2.