Recent biomarker search revealed that several mitochondrial proteins are detectable in the plasma and their over-expression is considered as potential biomarkers for diagnosis/prognosis of certain cancers (8, 9). Also, mitochondrial proteins are considered as targets for cancer therapy (7).AIFM3 is a mitochondrial protein that consists of 598 amino acids, with a molecular weight of 66 kDa (10). AIFM3 has 35% similarity with the sequence of the apoptosis-inducing factor (AIF). Similarly, AIFM3 has the ability to induce apoptosis (10,11). Nonetheless, analysis of mitochondrial proteome indicated that AIFM3 was overexpressed in CCA tissues in comparison with the matched adjacent non-cancerous tissues (12). Subsequently, we reported that higher serum AIFM3 levels were significantly associated with lymph node metastasis and shorter overall survival of patients with CCA, and AIFM3 could be an independent prognostic marker for patients with CCA (13). In this study, to elucidate the possible role of AIFM3 and the related signaling pathways in CCA cells, we used a combination of gene-silencing, mass spectrometry, and bioinformatic tools. Furthermore, using CCA cells, we examined the effects of AIFM3 gene knock-down on tumor cell motilities by migration-invasion assays. Moreover, AIFM3 expression levels in surgical specimens were used to validate its diagnostic/prognostic role.