The p14/p19 (ARF) (ARF) tumor suppressor gene is frequently mutated in human cancer. Recently ARF has been shown to localize to mitochondria and to induce autophagy. However the controls that regulate the trafficking of ARF to mitochondria remain unknown. We recently reported that 2-phenylethynesulfonamide (PES) selectively interacts with cytosolic heat shock protein 70 (HSP70) and inhibits its function; we further showed that PES promotes the death of tumor cells, and that this is associated with an impairment of lysosome function and an inhibition of autophagy. In the present work we used a mass spectrometry-based approach to identify mitochondrial ARF-binding proteins. We report that mitochondrial ARF interacts with HSP70. We show that treatment of cells with PES blocks the trafficking of ARF to mitochondria, indicating that interaction with HSP70 mediates the mitochondrial localization of ARF. We also show that PES inhibits the ability of ARF to induce autophagy, supporting the premise that localization to this organelle is critical for ARF-induced autophagy. Finally, we report that cells expressing high levels of ARF are more sensitive to PES than counterparts with ARF silenced. High levels of ARF are characteristic of tumor cells with enhanced MAPK signaling and advanced stage; therefore, these data support the premise that PES may show preferential cytotoxicity to advanced stage cancers.