The c-jun N-terminal kinases (JNKs) are responsive to stress stimuli leading to activation of proapoptotic proteins and transcription. Additionally, JNK mitochondrial localization has been reported. To selectively target mitochondrial JNK signaling, we exploited JNK interaction with its mitochondrial scaffold, Sab, using small interfering RNAs (siRNAs) and a cell-permeable peptide corresponding to the KIM1 domain of Sab. Gene silencing and peptide interference of this interaction disrupted JNK translocation to the mitochondria and reduced phosphorylation of Bcl-2 without significant impact on c-Jun phosphorylation or AP-1 transcription. In contrast, the JNK inhibitory peptide (TI-JIP1) prevented these three functions. Tat-Sab(KIM1) selectivity was also demonstrated in anisomycin-stressed HeLa cells where Tat-Sab(KIM1) prevented Bcl-2 phosphorylation, cell death, loss of mitochondrial membrane potential, and superoxide generation but not c-Jun phosphorylation. Conversely, TI-JIP1 prevented all aforementioned stress-induced events. This probe introduces a means to evaluate JNK-mediated events on the mitochondria without intervening in nuclear functions of JNK.